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RECONSTITUTION IN CELL-FREE EXTRACTS
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30 1.0
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FIo. 3. Membrane fusion measured by fluorescent dequenching. R 18-labeled dense endosomes were incubated with 2 mM vanadium-free ATP and amounts of lysosomes (r-I) or naitochondria (*), as shown (nag protein/incubation).
[8] N u c l e a r E n v e l o p e A s s e m b l y f o l l o w i n g M i t o s i s By RUPERT PFALLER a n d JOHN W . NEWPORT Entry into mitosis of higher eukaryotic cells is accompanied by a series of reversible morphological rearrangements that are indispensible to achieve cell duplication. The most prominent among them includes chromosome condensation, spindle formation, and breakdown of the cell nucleus. All the cellular components recruited to perform mitosis-specific functions are recycled at the end of mitosis, resulting in the reformation of interphase cells. In recent years in vitro systems have been developed to investigate these cell functions on a molecular level. One of the most valuable systems to study the cell cycle in vitro is derived from unfertilized eggs from Xenopus laevis frogs. Depending on the way these extracts are prepared, they can be METHODS IN ENZYMOLOGY,VOL 219
~ t © 1992by AcademicPre~ Inc. Allrights of~l~oductioninauy formreserved.
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used for the in vitro study of nuclear assembly (interphase extracts), ~ nuclear disassembly (mitotic extracts),2 or to perform several rounds of nuclear assembly and disassembly (cycling extracts)? Here we describe preparation and use of interphase extracts from Xenopus eggs that promote assembly of nuclei around a chromatin substrate and, therefore, constitute the experimental basis to study nuclear architecture, function, and dynamics on a molecular level. We will focus on membrane assembly of the nuclear envelope although it should be noted that the potential of Xenopus extracts has also been exploited in investigating structure, function, and dynamics of the nuclear pore complex and the nuclear lamina, the other components of the nuclear envelope. Fractionation of Xenopus laevis Eggs Progesterone-stimulated maturation induces oocytes to proceed from prophase of meiosis I to metaphase of meiosis II. At this stage the mature oocytes appear as unfertilized eggs after passing down the oviduct of the frog. Unfertilized Xenopus eggs are arrested in the second metaphase of meiosis by a calcium-sensitive activity called CSF (cytostatic factor). Cytostatic factor stabilizes the activity of the central cell cycle regulator MPF (M-phase promoting factor), a kinase specifically activated during mitosis. Activation of the eggs (either by using Ca2+-ionophores or breaking eggs in the absence of protein synthesis) leads to inactivation of MPF activity and extracts can be prepared that promote reconstitution of nuclei around a DNA substrate (interphase extracts). Extracts from nonactivated eggs, prepared in the presence of protein synthesis, retain their MPF activity and promote nuclear breakdown (mitotic extracts). Using Xenopus interphase extracts, distinct steps in the reformation of the cell nucleus at the end of mitosis can be resolved and studied in detail. The advantage of this system is that all the components are naturally present in the mitotic state and their transition into the interphase state during nuclear reconstitution can be investigated.
Hormone Induction of Unfertilized Xenopus laevis Eggs Hormone induction employing gonadotropin causes female Xenopus laevis frogs to lay eggs.4 This is done in two steps. In a first step, frogs are primed with pregnant mare serum gonadotropin. Two hundred units (U) i j. Newport,Cell48, 205 (1987). 2j. Newportand T. Spann, Cell48, 219 (1987). 3A. W. Murrayand M. W. Kirsehner,Nature(London) 339, 275 (1989). 4 j. Newportand M. Kirschner,Cell30, 675 (1982).
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of the hormone (Calbiochem, La JoUa, CA), dissolved in 0.5 ml water, is injected. In a second step, 2 - 7 days after priming, the frogs are injected with 500 U human chorionic gonadotropin (hCG) (Sigma, St. Louis, MO) the night before harvesting the eggs. Eggs are washed in fourfold-diluted MMR buffer [MMR contains 100 m M NaC1, 2 m M KC1, 1 m M MgC12, 2 m M CaCI2, 0.1 m M ethylene glycol-bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.8] and subsequently treated with a 2% (w/v) solution of cysteine, pH 7.8 (about 200 ml/amount of eggs laid by one frog) for about 5 min. During the cysteine treatment the jelly coat is removed and a tighter packing of the eggs occurs. The dejellied eggs are then washed quickly three times with MMR (200 ml each) to remove the cysteine and are transferrd onto a petri dish. Eggs that display irregular pigment distribution (the brown-colored pigment should be evenly concentrated on one-half of the surface of the eggs are removed under a light microscope using a Pasteur pipette. The eggs are then washed twice with 100 ml of lysis buffer (250 m M sucrose, 50 m M KCI, 2.5 m M MgC12, 10 m M HEPES, pH 7.4) containing 50/tg cycloheximide/ml. Cycloheximide is included to prevent any further protein synthesis, especially the cyclin subunit of MPF. 5 Finally, the eggs are transferred to a 15-ml Falcon tube (Becton Dickinson, Oxnard, CA) with dithiothreitol (DTT; 1 m M final concentration), the inhibitor of actin gelation cytochalasin B (5/zg/ml; Sigma), and a protease inhibitor mix containing aprotinin and leupeptin (10/zg/ml final concentration of each inhibitor; Sigma) are added. Eggs are packed at room temperature by gentle centrifugation for 15 sec at 100 g in a clinical centrifuge and excess buffer is removed. Nuclear assembly in vitro is dilution sensitive and, therefore, it is essential to attain a highly concentrated egg cytoplasm.
Preparation of Crude Egg Cytoplasm The packed eggs are crushed by centrifugation for 12 min at 12,000 g i n a Sorvall (Norwalk, CT) HB-4 rotor at 4 °. After centrifugation, three layers can be distinguished: a bottom layer containing yolk, a layer containing the wanted crude cytoplasmic fraction (crude interphase extract) in the middie, and a yellow layer of low density lipid on top (Fig. 1). The crude interphase extract is removed with a syringe. If supplemented with an ATP-regenerating system, it will support nuclear assembly around proteinfree DNA (for example, DNA from bacteriophage 2)2 These in vitro assembled nuclei are morphologically and functionally indistinguishable from intact, isolated nuclei. 5 A. W. Murray, M. J. Solomon, and M. W. Kirschner, Nature (London) 339, 280 (1989).
[8]
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NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
I hr
12 min
12,000 g
cytosol
200,000 g membranes
FIG. 1. Fractionation of eggs from Xenopus laevis, DejeUied eggs are crushed by centrifugation for 12 min at 12,000 g. The crude egg cytoplasm contained in the middle layer is removed and centrifuged for 1 hr at 200,000 g. From the cytoplasmic fractions separated in this step a cytosolic and a membrane fraction can be recovered that together promote in vitro assembly of nuclei around demembranated frog sperm chromatin.
Fractionation of Crude Interphase Extract Further fractionation of the crude interphase extract can be achieved by ultracentrifugation.6 The crude extract is supplemented with cytochalasin B (10 #g/ml) and aprotinin/leupeptin (10/~g/ml each) and spun for 75 min at 200,000 g at 4 ° in a Beckman (Palo Alto, CA) TLS 55 rotor. In case of large-scale preparations, centrifugation also can be carried out for 2 hr at 150,000 g at 4 ° in a Beckman SW 50.1 rotor. After ultracentrifugation the cytoplasmic fractions are separated from the cytosolic fraction according to their density (Fig. 1). The bottom layer consists predominantly of glycogen. On top of it there are two layers of membrane vesicles of different density (which can be distinguished by their different color) followed by the clear cytosolic fraction. Finally, there is a yellow layer of residual lipid. The lipid layer is carefully removed by suction. Then the clear cytosolic layer is removed and respun for 20 min at 200,000 g at 4 ° to remove residual particulate material. The resulting interphase cytosol is frozen in liquid nitrogen and kept at - 80 °, where it is stable for several months. The upper of the two membrane fractions is removed, diluted with 5-10 vol of lysis buffer and supplemented with aprotinin/leupeptin (10 #g/ml) and 1 m M DTT. It should be noted that only the upper mereK. L. Wilson and J. Newport, J. Cell BioL 107, 57 (1988).
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brane layer is required for nuclear reconstitution and the lower membrane layer may actually have inhibitory effects. The washed membranes are reisolated by sedimentation through 0.2 ml oflysis buffer containing 0.5 M sucrose (Beckman TLS 55 rotor, 20 min at 30,000 g, 4°). Finally, the membrane pellet is resuspended in lysis buffer containing 0.5 M sucrose in a final volume corresponding to 10% of the volume of the crude cytoplasmic fraction. Aliquots of 5- to 10-/tl volume are rapidly frozen in liquid nitrogen and stored at - 80°. Freezing of small aliquots of membranes has proved to be important to retain their ability for nuclear assembly.
Preparation of Mitotic Extracts Extracts from Xenopus eggs can be obtained under conditions that preserve MPF activity and keep it in a mitotic state. 2,7 Preparation of mitotic extracts occurs similar to interphase extracts, with the following changes. Eggs are washed in all steps with 0.1 M NaC1 instead of MMR. Before breaking the dejellied eggs, they are washed in threefold concentrated EB buffer (240 m M p-glycerophosphate, 60 m M MgC12, 80 m M EGTA, pH 7.3), which does not contain cycloheximide. Omission of cycloheximide is important to allow continuous protein synthesis, in particular synthesis of the cyclin subunit of MPF. After breaking the eggs by centrifugation, the crude mitotic extract is further fractionated by ultracentrifugation as described above. The MPF activity can be further enriched from mitotic extracts by a m m o n i u m sulfate precipitation. 7,s Mitotic cytosol is diluted with an equal volume of EB buffer (80 m M p-glycerophosphate, 15 m M MgC12, 20 m M EGTA, pH 7.3) containing 1 m M DTT and 0.5 m M ATP~,S and spun for 30 rain at 200,000 g and 4 ° in a Beckman TLS 55 rotor. To the supernatant, 0.43 vol of 3.6 M a m m o n i u m sulfate solution in EB buffer is added dropwise to give a final a m m o n i u m sulfate concentration of 1.08 M. After incubation on ice for 30 min, precipitated protein is sedimented by centrifugation (SorvaU HB-4 rotor, 10 min at 10,000 rpm and 4 o). Precipitated protein is dissolved in EB, 1 m M D T T , 0.5 mMATP~,S (in 20-30% of the volume of the undiluted cytosol) and dialyzed against 100 vol EB, 1 m M DTT, 0.1 m M ATPTS for 5 hr. The crude MPF preparation is frozen in liquid nitrogen and stored at - 8 0 °, at which it is stable for months. The MPF activity can be tested either by measuring phosphorylation ofhistone H 1, a specific substrate o f M P F kinase, 9,1°or measuring its ability to induce 7 W. G. Dunphy and J. W. Newport, J. CellBioL 106, 2047 (1988). s M. Wu and J. G. Gerhart, Dev. Biol. 79, 465 (1980). 9 D. Arion, L. Meijer, L. Brizuela, and D. Beach, Cell55, 371 (1988). l°W. G. Dunphy and J. W. Newport, Cell58, 181 (1989).
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NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
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nuclear envelope breakdown of in vitro assembled nuclei (see below). 7 Ammonium sulfate precipitation enriches MPF activity 5- to 10-fold and the yield is about 80%.
In Vitro Assembly of Nuclei Employing cytosol and membrane fractions isolated from Xenopus eggs in combination with a chromatin substrate, nuclei can be formed that, by morphological and functional criteria, are indistinguishable from isolated interphase nuclei. 6 They contain a double membrane, a nuclear lamina, and nuclear pore complexes? t The DNA is enclosed in the nuclear envelope in a decondensed form. These nuclei can perform DNA replication and specific transport through the nuclear pore complex. ~2 As already mentioned, when crude cytoplasm is used assembly of nuclei in vitro can also be achieved around protein-free DNA. The crude egg extract obviously contains all basic components necessary to form chromatin. To study specific aspects of nuclear envelope assembly, however, chromatin from demembranated X. laevis frog sperm is the preferred DNA substrate because cytosol and membrane fraction derived from crude interphase extracts are already sufficient to drive nuclear reconstitution.
Preparation of Demembranated Frog Sperm Chromatin A convenient and reliable chromatin substrate for nuclear envelope assembly is demembranated frog sperm chromatin. The following procedure is described for isolation of sperm chromatin from the testes of one frog.6,~3 Testes are removed from an adult male X. laevis frog and, after all surrounding tissue has been removed, they are put in l ml of buffer X [200 m M sucrose, 80 m M KCI, 15 m M NaC1, 5 m M ethylenediaminetetraacetic acid (EDTA), 7 m M MgC12, 15 m M piperazine-N,N'-bis(2-ethane sulfonic acid) (PIPES)-KOH, pH 7.2] on ice. To release the sperm, the testes are minced vigorously using tweezers. Isolation of the sperm is then achieved by repeated sedimentation and washing. First, big pieces of tissue are removed by centrifugation in a clinical centrifuge at 100 g for 10 sec. The supernatant containing released sperm is removed and kept on ice. To increase the yield, the pellet is reextracted with 0.5 ml buffer X as described above. The resulting supernatant is combined with the supernarant of the first extraction and centrifuged in a clinical centrifuge for 2 min ~l j. W. Newport, K. L. Wilson, and W. G. Dunphy, JCellBioL 111, 2247 (1990). 12 D. R. Finlay and D. J. Forbes, Cell66, 17 (1990). ~3 M. J. Lohka and Y. Masui, Science 220, 719 (1983).
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at 350 g at 4 ° to isolate the sperm. The sperm pellet is usually contaminated with some somatic and red blood cells, which sediment preferentially at the bottom of the pellet. Further purification, therefore, can be achieved by washing the crude sperm pellet, avoiding the tightly packed pellet of red blood cells at the bottom. The sedimented, white sperm are resuspended in I ml of buffer X, avoiding the reddish bottom pellet, transferred to new tubes, and sedimented by centrifugation as described above. The sperm should be washed this way at least three times. After the washing procedure is finished, the sperm are demembranated using lysolecithin. The sperm pellet is resuspended in 270/tl buffer X, warmed to room temperature, and 30/tl of a 0.5% (w/v) solution of lysolecithin (Sigma) in buffer X is added. The sperm then is suspended thoroughly and incubated at room temperature for 5 min. Then, 0.9 ml buffer X containing 3% (w/v) bovine serum albumin (BSA) is added to neutralize the lysolecithin. The demembranated sperm chromatin is reisolated by centrifugation for 2 min at 350 g at 4 ° and washed once in 1.2 ml buffer X containing 3% (w/v) BSA. The chromatin is then resuspended in 50/ll buffer X. The concentration of sperm chromatin is determined by counting the number of sperm contained in a 100-fold diluted aliquot using a hemacytometer. Aliquots of 5 - 10/tl (at a concentration of 50,000 sperm//A) are frozen in liquid nitrogen and stored at - 7 0 °. Successful removal of the sperm membrane is checked by combined light and fluorescence microscopy employing the DNA-specific fluorescence dye Hoechst 33258 (Hoechst-Roussel Pharmaceuticals, Somerville, NJ). Only chromatin molecules that no longer have an intact membrane will bind the dye and can be visualized. They can, therefore, be distinguished from intact sperm molecules, which can be detected by light microscopy. Treatment with lysolecithin usually yields more than 95% demembranated sperm chromatin.
Assembly of Synthetic Nuclei A reaction mixture for nuclear reconstitution contains interphase cytosol and interphase membranes, which can be used either from frozen stocks (as described above) or from freshly prepared extracts. 6 Demembranated frog sperm chromatin is used as DNA substrate from frozen stocks. Reconstitution of nuclei is strongly ATP dependent, therefore interphase cytosol is supplemented with an ATP-regenerating system consisting of 2 m M ATP, 10 m M creatine phosphate, and 50/lg creatine kinase/ml cytosol. ATP and creatine phosphate are added from 0.2 M stock solutions (in 10 m M potassium phosphate, adjusted to pH 7.0) and creatine kinase (Sigma) is added from 5-mg/ml stock solutions [dissolved
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in 50 m M NaC1, 50% (v/v) glycerol, 10 m M potassium phosphate, pH 7.0, and stored at -20°]. As already mentioned, nuclear assembly in vitro is sensitive to dilution of the cytosolic fraction, which will support formation of a nuclear envelope. Nuclear formation is completely blocked when the cytosol is diluted greater than twofold. A typical assay is composed of 100 #1 interphase cytosol supplemented with an ATP-regenerating system, 10#1 of interphase membranes, and demembranated frog sperm chromatin (typically 500-1000/#1 final concentration). Formation of a nuclear envelope occurs at room temperature and is followed by combined phase-contrast and fluorescence microscopy. For observation on a microscope equipped for light and fluorescence microscopy, 2 - 3 #1 of sample is added on a microscope slide to the same volume of the DNA-specific dye Hoechst 33258 (10/tg/ml), dissolved in 3.7% (v/v) formaldehyde (if fxation of the sample is required) or lysis buffer. The appropriate magnification for observing nuclei is about 250- to 400-fold. Typically during 1 - 2 hr of incubation the sperm chromatin becomes enclosed in a phase-dense nuclear envelope (visualized by light microscopy) and the DNA decondenses (visualized by fluorescence microscopy using the DNA dye Hoechst 33258) (Fig. 2). The size of the nuclei formed depends on the ratio of membrane versus chromatin concentration in the assay. At the membrane concentration used here the maximal size, expressed by the surface area of the nuclei formed, is about 400 #m2/nu cleus.~ It will decrease at concentrations of frog sperm chromatin higher than 2000/#1.
Steps of Nuclear Reconstitution Using the isolated fractions described above some specific steps of nuclear envelope assembly can be studied. For the Xenopus system, so far, the following steps have been characterized. Membrane vesicles derived from interphase extracts can bind to demembranated frog sperm chromatin in the absence of cytosolic components. 14,~5To allow membrane binding, however, the chromatin must be partially decondensed. Partial decondensation is promoted by nucleoplasmin ~4,~ (contained in a heat-stable cytosolic fraction) or functionally equivalent poly(L-glutamic acid).~5 With
14 j. W. Newport and W. G. Dunphy, J. Cell Biol. 116, 295 (1992). 15 R. Pfaller, C. Smythe, and J. W. Newport, Cellr5, 209 (1991). t6 A. Philipott, G. H. Leno, and R. A. Laskey, Cell6$, 569 0991).
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RECONSTITUTIO INNCELL-FREE EXTRACTS
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A
FIO. 2. Nuclear assembly in vitro. Assembly of nuclei in vitro was carried out as described in the text. The image of a reconstituted nucleus, visualiz~ by DNA-specific fluorescence microscopy (A) and phase-contrast microscopy (B) is shown. Bar, 3/zM.
an experimental system for the initial interaction of nuclear membrane vesicles with chromatin at hand, two aspects of nuclear envelope assembly can be investigated, namely subsequent steps in nuclear envelope assembly and regulation of these interactions during the course of the cell cycle. 1. Chromatin-bound membrane vesicles can be fused to form basic double-membrane structures on the chromatin surface in the presence of ATP and GTP. Membrane fusion is inhibited by N-ethylmaleimide and GTPyS, indicating mechanisms similar to membrane fusion in vesicular protein transport are involved.t4 Completion of nuclear envelope assembly from chromatin-bound membranes requires additional factors from interphase cytosol to allow assembly of nuclear pore complexes and the nuclear lamina. 2. Binding of membrane vesicles to chromatin is regulated by a phosphorylation regulatory system) s A regulatory kinase and phosphatase appear to be contained in interphase cytosol. Inhibition of the okadaic acidsensitive phosphatase of either type 1 or 2A leads to release of
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NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
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chromatin-bound membrane vesicles. Membrane release is caused by the regulatory kinase that, most likely, phosphorylates a membrane-localized component. In interphase extracts the equilibrium of activities of the regulatory kinase and phosphatase favors membrane binding. In mitotic extracts, this equilibrium is shifted toward membrane release and no requirement for okadaic acid is observed? 4
Controlled Decondensation of Frog Sperm Chromatin In sperm chromatin the DNA is organized in a highly condensed form. To observe membrane binding, the DNA must be partially decondensed. This can be achieved in either of two ways. The first approach employs heat-stable components of the cytosolic fraction? 4 Cytosolic proteins are denatured by heating at 95 ° for 5 - 10 min and sedimented by centrifugation for 20 min at 50,000 g at 4 ° in a Beckman rotor TLS 55. The protein components of the supernatant (termed "heat extract") consist predominantly of the three acidic proteins nucleoplasmin, N 1, and N2, which are involved in nucleosome assembly and chromosome decondensation. 17 In the heat extract, a partial decondensation of sperm chromatin can be achieved corresponding to a 25- to 30-fold volume increase. The extent of decondensation is dependent on the chromatin concentration and is maximal below a concentration of 3000 DNA molecules/#l heat extract. In the second approach, poly(L-glutamic acid) is substituted for the heat extract? 5 Nucleoplasmin contains tracts of polyglutamic acid that are thought to be functionally involved in chromatin decondensation? s Using commercially available poly(L-glutamic acid) (Sigma; average Mr 13 K) at concentrations of 0.5-2.0 mg/ml results in efficient, partial decondensation of sperm chromatin. Sperm chromatin decondensed by polyglutamic acid provides a defined system to investigate the interaction of nuclear envelope membranes with chromatin in vitro.
Formation of Nuclear Envelopefrom Membrane Vesicle~"Bound to Frog Sperm Chromatin When demembranated frog sperm chromatin (1000//11) is incubated in 100/A heat extract containing 10/~1 added membrane suspension (about 150/~g protein), the membranes bind to the decondensed sperm. After incubation for 1 hr at room temperature the sample is layered in a 0.6-ml borosilicate culture tube (Kimble, Vineland, N J) on top of I ml lysis buffer ,7 R. A. Laskey, B. M. Honda, A. D. Mills, and J. T. Finch, Nature (London) 275, 416 (1978). 18C. Dingwall, S. M. Dilworth, S. J. Black, S. E. Kearsey, L. S. Cox, and R. A. Laskey, EMBO J. 6, 69 (1987).
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containing 1 Msucrose and centrifuged for 5 min in a clinical centrifuge at 500 g at 4 o. Under these conditions, chromatin containing bound membrane vesicles is pelleted on the bottom and separated from unbound membranes. The resulting pellet is gently resuspended in 250-400/zl of interphase cytosol containing an ATP-regenerating system and incubated at room temperature for 1 hr. The formation of a phase-dense nuclear envelope is then assessed by combined light and fluorescence microscopy as described above.
Reversible Binding of Nuclear Membrane Vesicles to Frog Sperm Chromatin Binding of Membrane Vesicles to Frog Sperm Chromatin. To a solution of 180/zl poly(L-glutamic acid) (0.5 mg/ml) in lysis buffer demembranated frog sperm chromatin (1500//A) and membranes (0.3 mg/ml final protein concentration) are added. ATP and GTP~,S (Boehringer Mannheim, Indianapolis, IN) are added to 2 and 0.1 mMfinal concentration, respectively. GTP~,S, dissolved in 0.1 M HEPES, pH 7.2, containing 1 m M DTT, is added from a 20 m M stock solution. After incubation for 30 rain at room temperature, binding of membrane vesicles to chrornatin is assessed by fluorescence microscopy employing a combination of DNA-specific and membrane-specific fluorescence dyes. Hoechst 33258 is used to stain DNA (see above) and 6,6'-dihexylcarbocyanine (Kodak, Rochester, NY) (2/~g/ ml final concentration) to stain membranes. Both dyes are dissolved together in 3.7% formaldehyde and samples for microscopy are prepared by adding 2-3/A of sample to the same volume of dye solution on a microscope slide. Samples are best observed at about 300- to 600-fold magnification. Chromatin molecules, densely packed with bound membrane vesicles, can be easily distinguished from background fluorescence caused by unbound membrane vesicles (see Fig. 3). Release of Chromatin-Bound Membrane Vesicles Employing Okadaic Acid and Interphase Cytosol. Aliquots (30/tl) are mixed with 5/zl interphase cytosol containing an ATP-regenerating system and 0.5 mMGTP~,S. Okadaic acid (Moana Bioproducts, Honolulu, Hawaii), dissolved in H20, is added from a 0.1 m M stock solution to a final concentration of 1/tM. Relatively high concentrations of GTP~,S are required to inhibit fusogenic activities in interphase cytosol. To demonstrate that membrane release occurs as a result of the inhibition of a cytosolic protein phosphatase by okadaic acid, in one control sample only interphase cytosol and no okadaic acid is added and in a second control sample, which contains okadaic acid, lysis buffer is added instead of the interphase cytosol. Binding of membranes to chromatin is assessed by fluorscence microscopy as described
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FIG. 3. Reversible binding of membrane vesicles to frog sperm chromatin. As outlined in the text, membrane vesicles derived from Xenopus laevis egg extracts are first bound to demembranated frog sperm chromatin that was partially decondensed by poly(L-glutamic acid). Then aliquots of this substrate of chromatin-bound membrane vesicles are combined with okadaic acid (A), interphase cytosol (B), and both interphase cytosol and okadalc acid (C), respectively. At different time points, aliquots are removed and binding of membranes to chromatin is assessed by fluorescence microscopy employing fluorescence dyes specific for DNA (left) and membranes (right), respectively. After incubation for 1 hr, in the sample containing the combination of interphase cytosol and okadalc acid, the characteristic staining of DNA-associated membranes has disappeared due to release to chromatin-bound vesicles.
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above. Release of chromatin-bound membranes occurs within 30 to 60 min after addition in the sample containing interphase cytosol and okadaic acid while neither interphase cytosol nor okadaic acid alone affect membrane binding (Fig. 3). Depending on the quality, interphase cytosol can be diluted up to 20-fold and still promote membrane release within 1 hr. Membrane Release Employing Mitotic Extracts. Binding of membrane vesicles is carded out as described above, except EB buffer is used instead of lysis buffer to preserve MPF activity. After incubation for 30 min at room temperature, 5 ~I of mitotic extract, fractionated and enriched for MPF activity by ammonium sulfate precipitation (see above), is added to a 30-/~1 aliquot of the binding reaction. Release of membrane vesicles bound to chromatin is assessed by combined fluorescence microscopy for membranes and DNA, as described above. To achieve membrane release within 1 hr, the MPF-containing protein fraction can be diluted about 20-fold. Dependence of the observed membrane release on the mitotic state of the added protein fraction can be demonstrated by employing an ammonium sulfate-precipitated protein fraction isolated from interphase cytosol instead of mitotic cytosol. Although this protein fraction does not lead to release of chromatin-bound membrane vesicles by itself, it will in combination with okadaic acid, thus demonstrating that it contains both the regulatory kinase and phosphatase.
[9] F o r m a t i o n
of Endoplasmic Reticulum Networks in Vitro
By JAMES M . M C I L V A I N , JR., a n d M I C H A E L P . SHEETZ
Introduction The endoplasmic reticulum (ER) is an abundant tubulovesicular or planar membrane system that comprises approximately 50% of the total membrane content of a cell. Because of the density of ER membranes in the cytoplasm, it is difficult to visualize individual strands of ER except in the peripheral regions of highly spread cells. The peripheral ER strands in living cells are highly dynamic.',2 This supports the dynamic functions that have been proposed for the ER, i.e., the processing of exported molecules, 1C. Lee and L. B. Chen, Cell54, 37 (1988). 2 C. Lee, M. Ferguson, and L. B. Chen, J. CellBiol. 109, 2045 (1989).
METHODS 1N ENZYMOLOGY,VOL. 219
Copy6~ht© 1992by AcademicPress,Inc. All rightsof reproductionin any formreserved.
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30 1.0
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FIo. 3. Membrane fusion measured by fluorescent dequenching. R 18-labeled dense endosomes were incubated with 2 mM vanadium-free ATP and amounts of lysosomes (r-I) or naitochondria (*), as shown (nag protein/incubation).
[8] N u c l e a r E n v e l o p e A s s e m b l y f o l l o w i n g M i t o s i s By RUPERT PFALLER a n d JOHN W . NEWPORT Entry into mitosis of higher eukaryotic cells is accompanied by a series of reversible morphological rearrangements that are indispensible to achieve cell duplication. The most prominent among them includes chromosome condensation, spindle formation, and breakdown of the cell nucleus. All the cellular components recruited to perform mitosis-specific functions are recycled at the end of mitosis, resulting in the reformation of interphase cells. In recent years in vitro systems have been developed to investigate these cell functions on a molecular level. One of the most valuable systems to study the cell cycle in vitro is derived from unfertilized eggs from Xenopus laevis frogs. Depending on the way these extracts are prepared, they can be METHODS IN ENZYMOLOGY,VOL 219
~ t © 1992by AcademicPre~ Inc. Allrights of~l~oductioninauy formreserved.
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used for the in vitro study of nuclear assembly (interphase extracts), ~ nuclear disassembly (mitotic extracts),2 or to perform several rounds of nuclear assembly and disassembly (cycling extracts)? Here we describe preparation and use of interphase extracts from Xenopus eggs that promote assembly of nuclei around a chromatin substrate and, therefore, constitute the experimental basis to study nuclear architecture, function, and dynamics on a molecular level. We will focus on membrane assembly of the nuclear envelope although it should be noted that the potential of Xenopus extracts has also been exploited in investigating structure, function, and dynamics of the nuclear pore complex and the nuclear lamina, the other components of the nuclear envelope. Fractionation of Xenopus laevis Eggs Progesterone-stimulated maturation induces oocytes to proceed from prophase of meiosis I to metaphase of meiosis II. At this stage the mature oocytes appear as unfertilized eggs after passing down the oviduct of the frog. Unfertilized Xenopus eggs are arrested in the second metaphase of meiosis by a calcium-sensitive activity called CSF (cytostatic factor). Cytostatic factor stabilizes the activity of the central cell cycle regulator MPF (M-phase promoting factor), a kinase specifically activated during mitosis. Activation of the eggs (either by using Ca2+-ionophores or breaking eggs in the absence of protein synthesis) leads to inactivation of MPF activity and extracts can be prepared that promote reconstitution of nuclei around a DNA substrate (interphase extracts). Extracts from nonactivated eggs, prepared in the presence of protein synthesis, retain their MPF activity and promote nuclear breakdown (mitotic extracts). Using Xenopus interphase extracts, distinct steps in the reformation of the cell nucleus at the end of mitosis can be resolved and studied in detail. The advantage of this system is that all the components are naturally present in the mitotic state and their transition into the interphase state during nuclear reconstitution can be investigated.
Hormone Induction of Unfertilized Xenopus laevis Eggs Hormone induction employing gonadotropin causes female Xenopus laevis frogs to lay eggs.4 This is done in two steps. In a first step, frogs are primed with pregnant mare serum gonadotropin. Two hundred units (U) i j. Newport,Cell48, 205 (1987). 2j. Newportand T. Spann, Cell48, 219 (1987). 3A. W. Murrayand M. W. Kirsehner,Nature(London) 339, 275 (1989). 4 j. Newportand M. Kirschner,Cell30, 675 (1982).
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of the hormone (Calbiochem, La JoUa, CA), dissolved in 0.5 ml water, is injected. In a second step, 2 - 7 days after priming, the frogs are injected with 500 U human chorionic gonadotropin (hCG) (Sigma, St. Louis, MO) the night before harvesting the eggs. Eggs are washed in fourfold-diluted MMR buffer [MMR contains 100 m M NaC1, 2 m M KC1, 1 m M MgC12, 2 m M CaCI2, 0.1 m M ethylene glycol-bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.8] and subsequently treated with a 2% (w/v) solution of cysteine, pH 7.8 (about 200 ml/amount of eggs laid by one frog) for about 5 min. During the cysteine treatment the jelly coat is removed and a tighter packing of the eggs occurs. The dejellied eggs are then washed quickly three times with MMR (200 ml each) to remove the cysteine and are transferrd onto a petri dish. Eggs that display irregular pigment distribution (the brown-colored pigment should be evenly concentrated on one-half of the surface of the eggs are removed under a light microscope using a Pasteur pipette. The eggs are then washed twice with 100 ml of lysis buffer (250 m M sucrose, 50 m M KCI, 2.5 m M MgC12, 10 m M HEPES, pH 7.4) containing 50/tg cycloheximide/ml. Cycloheximide is included to prevent any further protein synthesis, especially the cyclin subunit of MPF. 5 Finally, the eggs are transferred to a 15-ml Falcon tube (Becton Dickinson, Oxnard, CA) with dithiothreitol (DTT; 1 m M final concentration), the inhibitor of actin gelation cytochalasin B (5/zg/ml; Sigma), and a protease inhibitor mix containing aprotinin and leupeptin (10/zg/ml final concentration of each inhibitor; Sigma) are added. Eggs are packed at room temperature by gentle centrifugation for 15 sec at 100 g in a clinical centrifuge and excess buffer is removed. Nuclear assembly in vitro is dilution sensitive and, therefore, it is essential to attain a highly concentrated egg cytoplasm.
Preparation of Crude Egg Cytoplasm The packed eggs are crushed by centrifugation for 12 min at 12,000 g i n a Sorvall (Norwalk, CT) HB-4 rotor at 4 °. After centrifugation, three layers can be distinguished: a bottom layer containing yolk, a layer containing the wanted crude cytoplasmic fraction (crude interphase extract) in the middie, and a yellow layer of low density lipid on top (Fig. 1). The crude interphase extract is removed with a syringe. If supplemented with an ATP-regenerating system, it will support nuclear assembly around proteinfree DNA (for example, DNA from bacteriophage 2)2 These in vitro assembled nuclei are morphologically and functionally indistinguishable from intact, isolated nuclei. 5 A. W. Murray, M. J. Solomon, and M. W. Kirschner, Nature (London) 339, 280 (1989).
[8]
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NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
I hr
12 min
12,000 g
cytosol
200,000 g membranes
FIG. 1. Fractionation of eggs from Xenopus laevis, DejeUied eggs are crushed by centrifugation for 12 min at 12,000 g. The crude egg cytoplasm contained in the middle layer is removed and centrifuged for 1 hr at 200,000 g. From the cytoplasmic fractions separated in this step a cytosolic and a membrane fraction can be recovered that together promote in vitro assembly of nuclei around demembranated frog sperm chromatin.
Fractionation of Crude Interphase Extract Further fractionation of the crude interphase extract can be achieved by ultracentrifugation.6 The crude extract is supplemented with cytochalasin B (10 #g/ml) and aprotinin/leupeptin (10/~g/ml each) and spun for 75 min at 200,000 g at 4 ° in a Beckman (Palo Alto, CA) TLS 55 rotor. In case of large-scale preparations, centrifugation also can be carried out for 2 hr at 150,000 g at 4 ° in a Beckman SW 50.1 rotor. After ultracentrifugation the cytoplasmic fractions are separated from the cytosolic fraction according to their density (Fig. 1). The bottom layer consists predominantly of glycogen. On top of it there are two layers of membrane vesicles of different density (which can be distinguished by their different color) followed by the clear cytosolic fraction. Finally, there is a yellow layer of residual lipid. The lipid layer is carefully removed by suction. Then the clear cytosolic layer is removed and respun for 20 min at 200,000 g at 4 ° to remove residual particulate material. The resulting interphase cytosol is frozen in liquid nitrogen and kept at - 80 °, where it is stable for several months. The upper of the two membrane fractions is removed, diluted with 5-10 vol of lysis buffer and supplemented with aprotinin/leupeptin (10 #g/ml) and 1 m M DTT. It should be noted that only the upper mereK. L. Wilson and J. Newport, J. Cell BioL 107, 57 (1988).
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brane layer is required for nuclear reconstitution and the lower membrane layer may actually have inhibitory effects. The washed membranes are reisolated by sedimentation through 0.2 ml oflysis buffer containing 0.5 M sucrose (Beckman TLS 55 rotor, 20 min at 30,000 g, 4°). Finally, the membrane pellet is resuspended in lysis buffer containing 0.5 M sucrose in a final volume corresponding to 10% of the volume of the crude cytoplasmic fraction. Aliquots of 5- to 10-/tl volume are rapidly frozen in liquid nitrogen and stored at - 80°. Freezing of small aliquots of membranes has proved to be important to retain their ability for nuclear assembly.
Preparation of Mitotic Extracts Extracts from Xenopus eggs can be obtained under conditions that preserve MPF activity and keep it in a mitotic state. 2,7 Preparation of mitotic extracts occurs similar to interphase extracts, with the following changes. Eggs are washed in all steps with 0.1 M NaC1 instead of MMR. Before breaking the dejellied eggs, they are washed in threefold concentrated EB buffer (240 m M p-glycerophosphate, 60 m M MgC12, 80 m M EGTA, pH 7.3), which does not contain cycloheximide. Omission of cycloheximide is important to allow continuous protein synthesis, in particular synthesis of the cyclin subunit of MPF. After breaking the eggs by centrifugation, the crude mitotic extract is further fractionated by ultracentrifugation as described above. The MPF activity can be further enriched from mitotic extracts by a m m o n i u m sulfate precipitation. 7,s Mitotic cytosol is diluted with an equal volume of EB buffer (80 m M p-glycerophosphate, 15 m M MgC12, 20 m M EGTA, pH 7.3) containing 1 m M DTT and 0.5 m M ATP~,S and spun for 30 rain at 200,000 g and 4 ° in a Beckman TLS 55 rotor. To the supernatant, 0.43 vol of 3.6 M a m m o n i u m sulfate solution in EB buffer is added dropwise to give a final a m m o n i u m sulfate concentration of 1.08 M. After incubation on ice for 30 min, precipitated protein is sedimented by centrifugation (SorvaU HB-4 rotor, 10 min at 10,000 rpm and 4 o). Precipitated protein is dissolved in EB, 1 m M D T T , 0.5 mMATP~,S (in 20-30% of the volume of the undiluted cytosol) and dialyzed against 100 vol EB, 1 m M DTT, 0.1 m M ATPTS for 5 hr. The crude MPF preparation is frozen in liquid nitrogen and stored at - 8 0 °, at which it is stable for months. The MPF activity can be tested either by measuring phosphorylation ofhistone H 1, a specific substrate o f M P F kinase, 9,1°or measuring its ability to induce 7 W. G. Dunphy and J. W. Newport, J. CellBioL 106, 2047 (1988). s M. Wu and J. G. Gerhart, Dev. Biol. 79, 465 (1980). 9 D. Arion, L. Meijer, L. Brizuela, and D. Beach, Cell55, 371 (1988). l°W. G. Dunphy and J. W. Newport, Cell58, 181 (1989).
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NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
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nuclear envelope breakdown of in vitro assembled nuclei (see below). 7 Ammonium sulfate precipitation enriches MPF activity 5- to 10-fold and the yield is about 80%.
In Vitro Assembly of Nuclei Employing cytosol and membrane fractions isolated from Xenopus eggs in combination with a chromatin substrate, nuclei can be formed that, by morphological and functional criteria, are indistinguishable from isolated interphase nuclei. 6 They contain a double membrane, a nuclear lamina, and nuclear pore complexes? t The DNA is enclosed in the nuclear envelope in a decondensed form. These nuclei can perform DNA replication and specific transport through the nuclear pore complex. ~2 As already mentioned, when crude cytoplasm is used assembly of nuclei in vitro can also be achieved around protein-free DNA. The crude egg extract obviously contains all basic components necessary to form chromatin. To study specific aspects of nuclear envelope assembly, however, chromatin from demembranated X. laevis frog sperm is the preferred DNA substrate because cytosol and membrane fraction derived from crude interphase extracts are already sufficient to drive nuclear reconstitution.
Preparation of Demembranated Frog Sperm Chromatin A convenient and reliable chromatin substrate for nuclear envelope assembly is demembranated frog sperm chromatin. The following procedure is described for isolation of sperm chromatin from the testes of one frog.6,~3 Testes are removed from an adult male X. laevis frog and, after all surrounding tissue has been removed, they are put in l ml of buffer X [200 m M sucrose, 80 m M KCI, 15 m M NaC1, 5 m M ethylenediaminetetraacetic acid (EDTA), 7 m M MgC12, 15 m M piperazine-N,N'-bis(2-ethane sulfonic acid) (PIPES)-KOH, pH 7.2] on ice. To release the sperm, the testes are minced vigorously using tweezers. Isolation of the sperm is then achieved by repeated sedimentation and washing. First, big pieces of tissue are removed by centrifugation in a clinical centrifuge at 100 g for 10 sec. The supernatant containing released sperm is removed and kept on ice. To increase the yield, the pellet is reextracted with 0.5 ml buffer X as described above. The resulting supernatant is combined with the supernarant of the first extraction and centrifuged in a clinical centrifuge for 2 min ~l j. W. Newport, K. L. Wilson, and W. G. Dunphy, JCellBioL 111, 2247 (1990). 12 D. R. Finlay and D. J. Forbes, Cell66, 17 (1990). ~3 M. J. Lohka and Y. Masui, Science 220, 719 (1983).
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RECONSTITUTION IN CELL-FREE EXTRACTS
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at 350 g at 4 ° to isolate the sperm. The sperm pellet is usually contaminated with some somatic and red blood cells, which sediment preferentially at the bottom of the pellet. Further purification, therefore, can be achieved by washing the crude sperm pellet, avoiding the tightly packed pellet of red blood cells at the bottom. The sedimented, white sperm are resuspended in I ml of buffer X, avoiding the reddish bottom pellet, transferred to new tubes, and sedimented by centrifugation as described above. The sperm should be washed this way at least three times. After the washing procedure is finished, the sperm are demembranated using lysolecithin. The sperm pellet is resuspended in 270/tl buffer X, warmed to room temperature, and 30/tl of a 0.5% (w/v) solution of lysolecithin (Sigma) in buffer X is added. The sperm then is suspended thoroughly and incubated at room temperature for 5 min. Then, 0.9 ml buffer X containing 3% (w/v) bovine serum albumin (BSA) is added to neutralize the lysolecithin. The demembranated sperm chromatin is reisolated by centrifugation for 2 min at 350 g at 4 ° and washed once in 1.2 ml buffer X containing 3% (w/v) BSA. The chromatin is then resuspended in 50/ll buffer X. The concentration of sperm chromatin is determined by counting the number of sperm contained in a 100-fold diluted aliquot using a hemacytometer. Aliquots of 5 - 10/tl (at a concentration of 50,000 sperm//A) are frozen in liquid nitrogen and stored at - 7 0 °. Successful removal of the sperm membrane is checked by combined light and fluorescence microscopy employing the DNA-specific fluorescence dye Hoechst 33258 (Hoechst-Roussel Pharmaceuticals, Somerville, NJ). Only chromatin molecules that no longer have an intact membrane will bind the dye and can be visualized. They can, therefore, be distinguished from intact sperm molecules, which can be detected by light microscopy. Treatment with lysolecithin usually yields more than 95% demembranated sperm chromatin.
Assembly of Synthetic Nuclei A reaction mixture for nuclear reconstitution contains interphase cytosol and interphase membranes, which can be used either from frozen stocks (as described above) or from freshly prepared extracts. 6 Demembranated frog sperm chromatin is used as DNA substrate from frozen stocks. Reconstitution of nuclei is strongly ATP dependent, therefore interphase cytosol is supplemented with an ATP-regenerating system consisting of 2 m M ATP, 10 m M creatine phosphate, and 50/lg creatine kinase/ml cytosol. ATP and creatine phosphate are added from 0.2 M stock solutions (in 10 m M potassium phosphate, adjusted to pH 7.0) and creatine kinase (Sigma) is added from 5-mg/ml stock solutions [dissolved
[8]
NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
67
in 50 m M NaC1, 50% (v/v) glycerol, 10 m M potassium phosphate, pH 7.0, and stored at -20°]. As already mentioned, nuclear assembly in vitro is sensitive to dilution of the cytosolic fraction, which will support formation of a nuclear envelope. Nuclear formation is completely blocked when the cytosol is diluted greater than twofold. A typical assay is composed of 100 #1 interphase cytosol supplemented with an ATP-regenerating system, 10#1 of interphase membranes, and demembranated frog sperm chromatin (typically 500-1000/#1 final concentration). Formation of a nuclear envelope occurs at room temperature and is followed by combined phase-contrast and fluorescence microscopy. For observation on a microscope equipped for light and fluorescence microscopy, 2 - 3 #1 of sample is added on a microscope slide to the same volume of the DNA-specific dye Hoechst 33258 (10/tg/ml), dissolved in 3.7% (v/v) formaldehyde (if fxation of the sample is required) or lysis buffer. The appropriate magnification for observing nuclei is about 250- to 400-fold. Typically during 1 - 2 hr of incubation the sperm chromatin becomes enclosed in a phase-dense nuclear envelope (visualized by light microscopy) and the DNA decondenses (visualized by fluorescence microscopy using the DNA dye Hoechst 33258) (Fig. 2). The size of the nuclei formed depends on the ratio of membrane versus chromatin concentration in the assay. At the membrane concentration used here the maximal size, expressed by the surface area of the nuclei formed, is about 400 #m2/nu cleus.~ It will decrease at concentrations of frog sperm chromatin higher than 2000/#1.
Steps of Nuclear Reconstitution Using the isolated fractions described above some specific steps of nuclear envelope assembly can be studied. For the Xenopus system, so far, the following steps have been characterized. Membrane vesicles derived from interphase extracts can bind to demembranated frog sperm chromatin in the absence of cytosolic components. 14,~5To allow membrane binding, however, the chromatin must be partially decondensed. Partial decondensation is promoted by nucleoplasmin ~4,~ (contained in a heat-stable cytosolic fraction) or functionally equivalent poly(L-glutamic acid).~5 With
14 j. W. Newport and W. G. Dunphy, J. Cell Biol. 116, 295 (1992). 15 R. Pfaller, C. Smythe, and J. W. Newport, Cellr5, 209 (1991). t6 A. Philipott, G. H. Leno, and R. A. Laskey, Cell6$, 569 0991).
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RECONSTITUTIO INNCELL-FREE EXTRACTS
[8]
A
FIO. 2. Nuclear assembly in vitro. Assembly of nuclei in vitro was carried out as described in the text. The image of a reconstituted nucleus, visualiz~ by DNA-specific fluorescence microscopy (A) and phase-contrast microscopy (B) is shown. Bar, 3/zM.
an experimental system for the initial interaction of nuclear membrane vesicles with chromatin at hand, two aspects of nuclear envelope assembly can be investigated, namely subsequent steps in nuclear envelope assembly and regulation of these interactions during the course of the cell cycle. 1. Chromatin-bound membrane vesicles can be fused to form basic double-membrane structures on the chromatin surface in the presence of ATP and GTP. Membrane fusion is inhibited by N-ethylmaleimide and GTPyS, indicating mechanisms similar to membrane fusion in vesicular protein transport are involved.t4 Completion of nuclear envelope assembly from chromatin-bound membranes requires additional factors from interphase cytosol to allow assembly of nuclear pore complexes and the nuclear lamina. 2. Binding of membrane vesicles to chromatin is regulated by a phosphorylation regulatory system) s A regulatory kinase and phosphatase appear to be contained in interphase cytosol. Inhibition of the okadaic acidsensitive phosphatase of either type 1 or 2A leads to release of
[8]
NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
69
chromatin-bound membrane vesicles. Membrane release is caused by the regulatory kinase that, most likely, phosphorylates a membrane-localized component. In interphase extracts the equilibrium of activities of the regulatory kinase and phosphatase favors membrane binding. In mitotic extracts, this equilibrium is shifted toward membrane release and no requirement for okadaic acid is observed? 4
Controlled Decondensation of Frog Sperm Chromatin In sperm chromatin the DNA is organized in a highly condensed form. To observe membrane binding, the DNA must be partially decondensed. This can be achieved in either of two ways. The first approach employs heat-stable components of the cytosolic fraction? 4 Cytosolic proteins are denatured by heating at 95 ° for 5 - 10 min and sedimented by centrifugation for 20 min at 50,000 g at 4 ° in a Beckman rotor TLS 55. The protein components of the supernatant (termed "heat extract") consist predominantly of the three acidic proteins nucleoplasmin, N 1, and N2, which are involved in nucleosome assembly and chromosome decondensation. 17 In the heat extract, a partial decondensation of sperm chromatin can be achieved corresponding to a 25- to 30-fold volume increase. The extent of decondensation is dependent on the chromatin concentration and is maximal below a concentration of 3000 DNA molecules/#l heat extract. In the second approach, poly(L-glutamic acid) is substituted for the heat extract? 5 Nucleoplasmin contains tracts of polyglutamic acid that are thought to be functionally involved in chromatin decondensation? s Using commercially available poly(L-glutamic acid) (Sigma; average Mr 13 K) at concentrations of 0.5-2.0 mg/ml results in efficient, partial decondensation of sperm chromatin. Sperm chromatin decondensed by polyglutamic acid provides a defined system to investigate the interaction of nuclear envelope membranes with chromatin in vitro.
Formation of Nuclear Envelopefrom Membrane Vesicle~"Bound to Frog Sperm Chromatin When demembranated frog sperm chromatin (1000//11) is incubated in 100/A heat extract containing 10/~1 added membrane suspension (about 150/~g protein), the membranes bind to the decondensed sperm. After incubation for 1 hr at room temperature the sample is layered in a 0.6-ml borosilicate culture tube (Kimble, Vineland, N J) on top of I ml lysis buffer ,7 R. A. Laskey, B. M. Honda, A. D. Mills, and J. T. Finch, Nature (London) 275, 416 (1978). 18C. Dingwall, S. M. Dilworth, S. J. Black, S. E. Kearsey, L. S. Cox, and R. A. Laskey, EMBO J. 6, 69 (1987).
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RECONSTITUTION IN CELL-FREE EXTRACTS
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containing 1 Msucrose and centrifuged for 5 min in a clinical centrifuge at 500 g at 4 o. Under these conditions, chromatin containing bound membrane vesicles is pelleted on the bottom and separated from unbound membranes. The resulting pellet is gently resuspended in 250-400/zl of interphase cytosol containing an ATP-regenerating system and incubated at room temperature for 1 hr. The formation of a phase-dense nuclear envelope is then assessed by combined light and fluorescence microscopy as described above.
Reversible Binding of Nuclear Membrane Vesicles to Frog Sperm Chromatin Binding of Membrane Vesicles to Frog Sperm Chromatin. To a solution of 180/zl poly(L-glutamic acid) (0.5 mg/ml) in lysis buffer demembranated frog sperm chromatin (1500//A) and membranes (0.3 mg/ml final protein concentration) are added. ATP and GTP~,S (Boehringer Mannheim, Indianapolis, IN) are added to 2 and 0.1 mMfinal concentration, respectively. GTP~,S, dissolved in 0.1 M HEPES, pH 7.2, containing 1 m M DTT, is added from a 20 m M stock solution. After incubation for 30 rain at room temperature, binding of membrane vesicles to chrornatin is assessed by fluorescence microscopy employing a combination of DNA-specific and membrane-specific fluorescence dyes. Hoechst 33258 is used to stain DNA (see above) and 6,6'-dihexylcarbocyanine (Kodak, Rochester, NY) (2/~g/ ml final concentration) to stain membranes. Both dyes are dissolved together in 3.7% formaldehyde and samples for microscopy are prepared by adding 2-3/A of sample to the same volume of dye solution on a microscope slide. Samples are best observed at about 300- to 600-fold magnification. Chromatin molecules, densely packed with bound membrane vesicles, can be easily distinguished from background fluorescence caused by unbound membrane vesicles (see Fig. 3). Release of Chromatin-Bound Membrane Vesicles Employing Okadaic Acid and Interphase Cytosol. Aliquots (30/tl) are mixed with 5/zl interphase cytosol containing an ATP-regenerating system and 0.5 mMGTP~,S. Okadaic acid (Moana Bioproducts, Honolulu, Hawaii), dissolved in H20, is added from a 0.1 m M stock solution to a final concentration of 1/tM. Relatively high concentrations of GTP~,S are required to inhibit fusogenic activities in interphase cytosol. To demonstrate that membrane release occurs as a result of the inhibition of a cytosolic protein phosphatase by okadaic acid, in one control sample only interphase cytosol and no okadaic acid is added and in a second control sample, which contains okadaic acid, lysis buffer is added instead of the interphase cytosol. Binding of membranes to chromatin is assessed by fluorscence microscopy as described
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NUCLEAR ENVELOPE ASSEMBLY FOLLOWING MITOSIS
71
FIG. 3. Reversible binding of membrane vesicles to frog sperm chromatin. As outlined in the text, membrane vesicles derived from Xenopus laevis egg extracts are first bound to demembranated frog sperm chromatin that was partially decondensed by poly(L-glutamic acid). Then aliquots of this substrate of chromatin-bound membrane vesicles are combined with okadaic acid (A), interphase cytosol (B), and both interphase cytosol and okadalc acid (C), respectively. At different time points, aliquots are removed and binding of membranes to chromatin is assessed by fluorescence microscopy employing fluorescence dyes specific for DNA (left) and membranes (right), respectively. After incubation for 1 hr, in the sample containing the combination of interphase cytosol and okadalc acid, the characteristic staining of DNA-associated membranes has disappeared due to release to chromatin-bound vesicles.
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RECONSTITUTION IN CELL-FREE EXTRACTS
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above. Release of chromatin-bound membranes occurs within 30 to 60 min after addition in the sample containing interphase cytosol and okadaic acid while neither interphase cytosol nor okadaic acid alone affect membrane binding (Fig. 3). Depending on the quality, interphase cytosol can be diluted up to 20-fold and still promote membrane release within 1 hr. Membrane Release Employing Mitotic Extracts. Binding of membrane vesicles is carded out as described above, except EB buffer is used instead of lysis buffer to preserve MPF activity. After incubation for 30 min at room temperature, 5 ~I of mitotic extract, fractionated and enriched for MPF activity by ammonium sulfate precipitation (see above), is added to a 30-/~1 aliquot of the binding reaction. Release of membrane vesicles bound to chromatin is assessed by combined fluorescence microscopy for membranes and DNA, as described above. To achieve membrane release within 1 hr, the MPF-containing protein fraction can be diluted about 20-fold. Dependence of the observed membrane release on the mitotic state of the added protein fraction can be demonstrated by employing an ammonium sulfate-precipitated protein fraction isolated from interphase cytosol instead of mitotic cytosol. Although this protein fraction does not lead to release of chromatin-bound membrane vesicles by itself, it will in combination with okadaic acid, thus demonstrating that it contains both the regulatory kinase and phosphatase.
[9] F o r m a t i o n
of Endoplasmic Reticulum Networks in Vitro
By JAMES M . M C I L V A I N , JR., a n d M I C H A E L P . SHEETZ
Introduction The endoplasmic reticulum (ER) is an abundant tubulovesicular or planar membrane system that comprises approximately 50% of the total membrane content of a cell. Because of the density of ER membranes in the cytoplasm, it is difficult to visualize individual strands of ER except in the peripheral regions of highly spread cells. The peripheral ER strands in living cells are highly dynamic.',2 This supports the dynamic functions that have been proposed for the ER, i.e., the processing of exported molecules, 1C. Lee and L. B. Chen, Cell54, 37 (1988). 2 C. Lee, M. Ferguson, and L. B. Chen, J. CellBiol. 109, 2045 (1989).
METHODS 1N ENZYMOLOGY,VOL. 219
Copy6~ht© 1992by AcademicPress,Inc. All rightsof reproductionin any formreserved.
