Cell. Vol. 65, 209-217,

April 19. 1991. Copyright

0 1991 by Cell Press

Assembly/Disassembly of the Nuclear Envelope Membrane: Cell Cycle-Dependent Binding of Nuclear Membrane Vesicles to Chromatin In Vitro Rupert Pfaller, Carl Smythe, and John W. Newport Department of Biology, B-022 University of California, San Diego La Jolla, California 92093

Summary Dissociation and association of membranes with chromatln at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employlng purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhlbltors, that membrane-chromatln association Is regulated by a phosphataselklnase system. In interphase, the balance In thls system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin blnding. At mitosis the membrane receptor Is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatln. Rather, binding of membranes to chromatin at mitosis appears to be regulated Indirectly by MPF through Its action on a phosphataselklnase system that directly modulates the phosphorylation state of a nuclear membrane component.

allows the lamin proteins to repolymerize (Gerace and Blobel, 1980). Recently, we have presented evidence that suggests that in Xenopus embryos, the dephosphorylation of lamins at the end of mitosis occurs after the lamins are transported into nuclei (Newport et al., 1990). We have also presented evidence that the single Xenopus embryonic lamin Llll (Benavente et al., 1985; Stick and Hausen, 1985) is not required for either the binding of membrane vesicles to the surface of chromatin or the fusion of these bound vesicles to each other to form an intact nuclear envelope (Newport et al., 1990; Newport, submitted). Current evidence indicates that the binding of membrane vesicles to chromatin at the end of mitosis is mediated by the interaction of a membrane-bound receptor with a chromatin-bound ligand (Wilson and Newport, 1988). Clearly, the regulation of the reversible interaction of these two components with each other is an important aspect of nuclear envelope dynamics during mitosis. In this report, we have investigated one aspect of nuclear envelope dynamics, that is, how the association and dissociation of membranes tochromatin are regulated during mitosis. Using purified membrane vesicles, chromatin, and a fractionated cell-free system derived from Xenopus eggs, we have investigated whether membrane-chromatin association at mitosis is regulated by a phosphorylation/dephosphorylation regulatory system, and if so, how MPF kinase participates in this process. Our results indicate that at least one step in nuclear membrane assembly at mitosis is regulated by a kinaselphosphatase system that is distinct from, but regulated by, MPF kinase.

Introduction Results At the beginning of mitosis in almost all eukaryotic cells, the nuclear DNA dissociates from the nuclear envelope and condenses to form compact chromosomes. In eukaryotic cells that undergo an open mitosis, the nuclear envelope vesicularizes and the nuclear lamins depolymerize at this time in the cell cycle. At the completion of mitosis, these membrane vesicles and lamin proteins bind to the surface of condensed DNA and reestablish an intact nuclear envelope (for review, see Newport and Forbes, 1987; Gerace and Burke, 1988). Precisely how these processes are reversibly regulated at mitosis is still an open question. Work from several laboratories has provided convincing evidence that the reversible disassembly and reassembly of the nuclear lamina at the beginning and end of mitosis is regulated by phosphorylation (Gerace and Blobel, 1980; Miake-Lye and Kirschner, 1985). Furthermore, recent evidence strongly implicates the pivotal regulator of mitosis, MPF, as playing an active and direct role in the phosphorylation of the lamins within the nuclear envelope (Peter et al., 1990). Thus, it appears that lamin disassembly at the onset of mitosis occurs owing to the phosphorylation of two specific sites on the nuclear lamin protein by the MPF kinase (Ward and Kirschner, 1990; Heald and McKeon, 1990; Peter et al., 1990). At the completion of mitosis, dephosphorylation by an uncharacterized phosphatase

Binding of Nuclear Membranes to Chromatin In Vltro Is Dependent on Phosphorylatlon/Dephosphorylation In a previous investigation from our laboratory, a cytosol and membrane fraction isolated from crude Xenopus laevis egg interphase extracts were employed to form nuclei around demembranated Xenopus frog sperm chromatin (Wilson and Newport, 1988). Subsequently, we found that membranes purified from Xenopus eggs would bind to chromatin in the absence of cytosol (Newport, submitted). To further study the binding of nuclear membranes to chromatin, the first step of the nuclear assembly process, we developed the following binding assay. Nuclear membranes were isolated from crude Xenopus egg interphase cytoplasm by high speed centrifugation (Wilson and Newport, 1988). These membranes were employed in binding experiments with demembranated frog sperm chromatin. We found that when membranes and chromatin were incubated in the presence of polyglutamic acid, ATP, and GTPyS, binding of membrane vesicles to the chromatin could be observed by fluorescence microscopy employing the lipophillic dye 3,3’-dihexyloxacarbocyanine (DHCC; Figure 1A). GTPyS was required to prevent fusion of DNA-bound membranes (Newport, submitted).

Cell 210

DNA

memb.

A no add.

B +okad. acid

C +30%ASiph.

D

+okad. acid, +30%ASiph. Figure 1. Reversible Sperm Chromatin

Binding

of Nuclear

Membrane

Vesicles

to Frog

Nuclear membranevesicles were incubated with demembranated frog sperm chromatin in the presence of polyglutamic acid, ATP, and GTP$, as described in the Experimental Procedures. After incubation for 30 min at 22’C, 30 ~1 aliquots of the binding reaction were incubated either with (A) 5 pl of EB, (B) 5 pl of EB and okadaic acid (1 pM final concentration in the assay: okadaic acid was always added from an aqueous stock solution), (C) 5 pl of 30% AS-interphase, or(D) 5 ~1 of 30% AS-interphase and okadaic acid (1 PM final concentration in the assay). Membrane binding was assessed at different time points by fluorescence microscopy. DNA stain (left panels) and membrane stain (right panels) of individual chromatin molecules after 1 hr incubation are shown. Scale bar is 10 pm.

In initial binding experiments, chromatin swelling was achieved by using the supernatant of interphase cytosol heated to 95% (“heat extract”). This procedure denatured all cytosolic proteins except nucleoplasmin and two other proteins called Nl and N2 (Laskey et al., 1978). These three proteins are thought to be involved in nucleosome assembly and chromatin decondensation (Newport, submitted). They contain an unusually high content of acidic amino acids. In particular, they all contain tracts of polyglutamic acid (Dingwall et al., 1987). We have found that chromatin swelling can be induced in the absence of nucleoplasmin, Nl, and N2 if polyglutamic acid is added to the chromatin. Therefore, we have used polyglutamic acid and ATP to substitute for the heat extract, and thus have been able to investigate membrane binding to chromatin in a defined assay system consisting of isolated, washed membranes, demembranated sperm chromatin, and a buffer containing ATP, GTPyS, and polyglutamic acid. In the course of the cell cycle, the nuclear envelope membrane is vesicularized during mitosis and reconstituted during interphase, a process that requires reversible binding of nuclear membranes to the DNA. Precedents from other aspects of nuclear organization (see Introduction) led us to assume that phosphorylationldephosphorylation might be the key regulatory process in determining the equilibrium of membrane binding to chromatin. To investigate the possible involvement of a kinaselphosphatase regulatory system in controlling membrane binding to chromatin, we examined the effect of okadaic acid, an

inhibitor of phosphatases 1 and 2A (Cohen et al., 1990), on the binding of membranes to frog sperm chromatin. To do this, purified membrane vesicles were allowed to bind chromatin, as described above (binding reaction). When okadaic acid (1 PM final concentration) was added to the binding reaction, the membranes remained attached to chromatin (Figure 1 B). To test whether a regulatory kinasel phosphatase system might be present in the soluble fraction of Xenopus interphase extract, we added a preformed chromatin-membrane complex and okadaic acid to a cytosolic interphase fraction made from Xenopus eggs (Newport, 1987). Under these conditions we observed that the prebound membranes now dissociated from the chromatin (not shown). To further purify the soluble components that caused membrane release in the presence of okadaic acid, the soluble components of the interphase extract were fractionated by ammonium sulfate precipitation. We found that adding okadaic acid together with a 30% ammonium sulfate fraction isolated from Xenopus egg interphase cytosol (termed 30% AS-interphase) resulted in dissociation of membranes bound to chromatin (membrane release; Figure 1 D). In the absence of okadaic acid, the 30% AS-interphase displayed no membrane release activity (Figure 1C). Membrane release activity was dependent both on the concentration of okadaic acid and on the 30% AS-interphase concentration. Concentrations below 1 PM okadaic acid no longer induced membrane release. The 30% AS-interphase could be diluted up to lo-fold and still cause membrane release within 1 hr. Thus, these results show that by blocking phosphatase activity in an extract that normally promotes nuclear envelope assembly, we have activated one of the important steps in nuclear envelope disassembly, namely, dissociation of membranes from chromatin. To determine if the okadaic acid-sensitive phosphatase involved in membrane binding was of the 1 or 2A type, we employed a synthetic inhibitor peptide, highly specific for phosphatase 1 (Aitken and Cohen, 1982; generously provided by P. Cohen, University of Dundee), in the membrane release assay. Concentrations up to 3 PM of the inhibitor peptide did not promote membrane release, indicating a phosphatase of the 2A type to be involved in the regulation of membrane binding to chromatin. However, this question can only be answered unequivocally once the phosphatase has been purified. At present, we cannot exclude inactivation of the inhibitor peptide due to degradation in our rather crude assay reactions. The results presented above show that the inhibition of phosphatase activity in interphase extracts causes membranes to dissociate from chromatin. This suggests that a kinase-mediated phosphorylation step is involved in the release of nuclear envelope membranes from chromatin. By blocking phosphatases with okadaic acid, we may have stabilized this phosphorylation step, and as a result vesicles are released from chromatin. The role of a possible kinase in membrane release was investigated in two ways. In the first approach, we investigated whether release of membranes from chromatin was ATP-dependent. To do this, the 30% AS-interphase fraction was depleted of ATP

Assembly/Disassembly 211

of the Nuclear

Envelope

Membrane

that binding is under the control of a phosphataselkinase regulatory system, with dephosphorylation required for membrane binding and phosphorylation required for membrane release.

Figure 2. Binding of Nuclear Membrane Vesicles matin Is under Control of a PhosphataselKinase

to Frog Sperm ChroRegulatory System

Binding of nuclear membrane vesicles to frog sperm chromatin was carried out in three parallel samples, as described in the Experimental Procedures. After incubation for 20 min at 22OC, the binding reactions and, simultaneously, 30% AS-interphase were treated with either 50 U of hexokinase per ml and 1 mM glucose ([A], no ATP depletion), 50 U of hexokinase per ml and 20 mM glucose ([B], ATP depletion), or 5 mM P-aminopurine ([Cl, kinase inhibitor) for 30 min at 22OC. Then, 30 pl aliquots of each binding reaction were incubated with 5 ul of the corresponding 30% AS-interphase and 1 pm of okadaic acid. Vesicle binding to chromatin was assessed by fluorescence microscopy as described in Figure 1. DNA (left panels) and membrane stain (right panels) after 1 hr incubation are shown.

by treating it with hexokinase (50 U/ml) and glucose (20 mM) for 30 min (Newmeyer et al., 1966). In a separate reaction, membranes were bound to chromatin and then depleted of ATP by the hexokinase/glucose reaction. This chromatin-membrane complex was then added to the ATP-depleted extract in the presence of okadaic acid. Under these conditions, no membrane release from chromatin was observed (Figure 28). In a control experiment, hexo kinase treatment of the 30% AS-interphase was carried out in the presence of 1 mM glucose (leading to low ATP consumption; Newmeyer et al., 1966). When a chromatin-membrane substrate also treated with hexokinase and 1 mM glucose was added to this extract in the presence of okadaic acid, release of chromatin-bound membrane vesicles occurred (Figure 2A). The same results were obtained when unfractionated interphase cytosol was used instead of the 30% AS-interphase (not shown). These experiments demonstrate that the okadaic acid-dependent release of membranes from chromatin is ATP dependent. In a second approach, 2-aminopurine, a kinase inhibitor, was used to inhibit the putative kinase. To do this, 30% AS-interphase and chromatin-bound membranes were incubated separately with 5 mM P-aminopurine, then combined, and finally, okadaic acid was added. 2-aminopurine blocked release of chromatin-bound membrane vesicles (Figure 2C), in contrast to a control reaction lacking the kinase inhibitor (not shown). The same result was obtained with a different kinase inhibitor, fluorosulfonyl-benzoyl-5’adenosine (FSBA). In summary, we have established a visual in vitro assay for the specific binding of nuclear membrane vesicles to chromatin. Using reversibility of binding as a criterion for specificity, we have demonstrated

MPF Can Trigger the Release of Chromatin-Bound Membrane Vesicles The results presented above suggest that the binding of membrane to DNA is determined by the activities of a phosphataselkinase regulatory system. Reversible membrane binding to chromatin is one of the key reactions leading to assembly/disassembly of the nuclear envelope at mitosis. This suggests that the phosphatase/kinase system may be subject to regulation by mitotic regulators. To address this question, we took advantage of the observation that interphase extracts from Xenopus eggs can be converted into mitotic extracts by adding cyclin, a protein that forms a complex with the cdc2 subunit of MPF and, in a series of subsequent reactions that are currently only partly understood, leads to the formation of active MPF kinase (Murray and Kirschner, 1969; Minshull et al., 1990; Solomon et al., 1990; for a review, see Lewin, 1990). To activate MPF, we used a recombinant cyclin fusion protein consisting of a glutathione S-transferase moiety fused to an N-terminally truncated cyclin Bl from sea urchin (a generous gift from M. Solomon and M. Kirschner, UCSF). After overexpression in Escherichiacoli, purification of this fusion protein is achieved by affinity chromatography on glutathione Sepharose (Solomon et al., 1990). When added to Xenopus egg interphase cytosol, the cyclin construct activated MPF, as judged by measuring histone Hl kinase activity (Figure 3C). Furthermore, we alsoobserved release of chromatin-bound membrane vesicles when cyclin was added to an interphase cytosol (Figure 38). The cyclin construct by itself displayed neither histone Hl kinase activity nor membrane release activity (Figure 3A). Phosphorylation by MPF Klnase Does Not Lead Directly to Membrane Release The observation that MPF caused membrane release even in the absence of okadaic acid raised the possibility that cdc2 kinase activity was directly involved in the phosphorylation that causes membranes to be released from chromatin. Several lines of evidence indicate that this is not the case. In a first approach, we investigated directly whether cdc2 kinase contained membrane release activity. To do this, the purified cyclin construct was added to interphase cytosol to form the cdc2-cyclin complex active as MPF. MPF was then purified from this extract by affinity chromatography, exploiting the ability of the glutathione S-transferase moiety of the cyclin construct to bind to glutathione Sepharose. MPF eluted from the affinity column was active, based on both its histone Hl kinase activity (Figure 4A) and its ability to induce nuclear envelope breakdown when added to an interphase extract (not shown). However, when this active, purified MPF was added directly to a complex of chromatin-bound membranes, it failed to release the membranes (Figures 48 and 4C, left panels). Only in combination with the 30% AS-interphase fraction (which could also be replaced by

Cell 212

interphase cyclin Figure 3. Membrane Conditions

oRelease

+ Activity

+ -

I

,

MPF no 30% AS-iph. Is Stimulated

under

MPF + 30% AS-iph.

M-Phase

Nuclear membrane vesicles were bound to chromatin (see the Experimental Procedures). In parallel, affinity-purified cyclin construct (3 mg of protein per ml)was incubated for 20 min at 22OC in a IO-fold dilution with either EB or interphase cytosol (MPF activation). Then, 30 ul aliquots of the binding reaction were incubated with 5 pl of either (A) the cyclin-EB mixture or(B) cyclin-interphase mixture. Release of membranes bound to chromatin was followed, as described in Figure I. In parallel, (C) histone Hl kinase assays of the cyclin construct alone, interphase cytosol alone, and cyclin construct combined with interphase cytosol were carried out. The samples were incubated for 20 min at 22OC and assayed for histone Hl kinase activity in a 1OO-fold dilution.

interphase cytosol) did purified MPF cause release of membrane vesicles from chromatin (Figures 46 and 4C, right panels). To further investigate whether the MPF kinase was acting directly to cause membrane release from chromatin, we examined whether the c&2 subunit of MPF was present in the 30% AS-interphase, using Western blots probed with anti-c&2 antibody. As shown above, this 30% fraction contains the kinase activity, causing release of membranes from chromatin in the presence of okadaic acid (Figure 1). However, Western blot analysis demonstrated that all of the cdc2 protein was present in a 30-660/o ASfraction, and no c&2 protein was present in the O-30% AS-fraction (Figure 5). Consistent with this finding, neither membrane release activity nor histone Hl kinase activity was generated in the 30% AS-interphase fraction upon addition of the cyclin construct. Together, these results show that although the cdc2 kinase can cause membrane release from chromatin, the kinase is not directly involved in the phosphorylation that is ultimately responsible for membrane dissociation. Sequence analysis has revealed that there are two closely related forms of c&2 present in Xenopus laevis (M. Philippe, personal communication; Milarski et al., submitted). While one form (termed c&P-A and assayed in Figure 5) is the catalytic subunit of MPF kinase, the function of the other form (termed c&P-B) is currently not well

Figure tivity

4. Purified

MPF by Itself Contains

No Membrane

Release

Ac-

MPF was purified by affinity chromatography as described in the Experimental Procedures. EB and 30% AS-interphase were each mixed with an equal volume of purified MPF, incubated for 20 min at 22OC, and then assayed for histone Hl kinase activity or membrane release activity. (A) Histone Hl kinase activity: the samples were assayed in a IO-fold dilution. Background activity of 30% AS-interphase in the absence of MPF was subtracted. (B) and (C) Membrane release assays: 30 pl aliquots of a binding reaction were added to 10 ul of MPF that had been incubated either in EB (left panels) or interphase cytosol (right panels), as described above. Release of chromatin-bound membrane vesicles was followed by fluorescence microscopy. DNA stain (B) and membrane stain (C) after incubation for 100 min at 22OC is shown.

understood. Using a C-terminus-specific antibody against c&2-B, we found that the 30% AS-interphase contained about 30% of the c&2-B present in unfractionated interphaseextracts.Toruleoutthepossibilitythatapotential kinase activity associated with c&2-B causes membrane release, we selectively removed both cdcP-A and cdcSB from interphase cytosol. To do this, we took advantage of the fact that both of these two cdc2 proteins bind to the product of the sucl gene from Saccharomyces pombe, ~13 (Brizuela et al., 1967; Dunphy et al., 1968). Recombinant ~13 was purified from overexpressing E. coli cells and immobilized on CNBr-activated Sepharose (~13 beads) to form an affinity matrix for cdc2-A and cdcP-B (Dunphy et al., 1988). We used ~13 beads to deplete both proteins from Xenopus interphase cytosol. The extent of affinity depletion of cdc2 was monitored by immunoblotting (Figure 6A). Using ~13 chromatography, we could deplete cdc2-A and cdc2-B from interphase cytosol almost quantitatively (more than 95%; Figure 6A). When okadaic acid and a chromatin-membrane substrate were added to a cdc2-A- and cdc2-B-depleted extract, the membranes dissociated from the chromatin (Figure 6B). At the highest possible dilution of interphase extracts, release of the membranes occurred at the same rate both in cdc2depleted extracts and in nondepleted extracts. Therefore,

A$mbly/Disassembly

of the Nuclear

Envelope

Membrane

A Pl3 depletion AS-iph.

30%

B

30-66%

Figure 5. cdc2 Is Highly Depleted in the 30% AS-Interphase Fraction Containing Membrane Release Activity Interphase cytosol was fractionated with ammonium sulfate as described in the Experimental Procedures. In a first step, ammonium sulfate was added to 36% (v/v) final concentration, and precipitated protein was isolated by centrifugation (30% AS-interphase). In a second step, the supernatant was adjusted to 66% (v/v) final concentration and, after incubation for 30 min on ice, precipitated protein was recovered by centrifugation (3646% AS-interphase). Both protein pellets were dissolved in EB and dialyzed against 100 vol of the same buffer. Equivalent volumes of both fractions were analyzed for their cdc2 content by immunoblotting. Lane 1: 30% AS-interphase. Lane 2: 3666% AS-interphase.

depletion

of c&2-A

nase required

and

cdcbB

does not remove the ki-

for membrane release Taken together, these results suggest not directly involved in the phosphorylation

from chromatin. that MPF kinase is event causing

release of chromatin-bound membrane vesicles. Instead, MPF appears to be involved in the activation of the regulatory enzymes that eventually bring about membrane dissociation. The Substrate for the Phosphorylatlon Regulatory System Is Located on the Membrane Vesicles The results presented above indicate that a soluble kinasel phosphatase system is involved in modulating membrane binding to chromatin. Our result6 are consistent with a model whereby phosphorylation of a protein leads to vesicle release, whereas dephosphorylation of the same protein causes vesicles to associate with chromatin. The protein target modified by this kinaselphosphatase system could be located on either chromatin or the membrane vesicles. To distinguish between these two possibilities, purified membranes were incubated in interphase extract containing an ATP-regenerating system, either in the presence or absence of okadaic acid. Following a 30 min incubation, membranes were recovered by centrifugation, washed once, and resuspended. If the target of the kinasel phosphatase system was located on the membranes, then we would expect this target to be phosphorylated in extracts containing okadaic acid and to become dephosphorylated in extracts lacking okadaic acid. Therefore, the recovered and washed membranes isolated from the extract lacking okadaic acid should bind chromatin, while membranes recovered from extracts containing okadaic acid should no longer bind chromatin. When each of the membrane fractions were tested for binding activity to chromatin in buffer, we found that membranes incubated in interphase cytosol alone bound chromatin, while membranes incubated in cytosol containing okadaic acid did not bind. This result suggests that one target for the ki-

+

-

DNA

memb.

Figure 6. MPF Kinase Is Not Directly Involved in the Membrane Release Reaction (A) cdc2A and cdc2-6 were depleted from interphase cytosol by binding to p13 beads as described in the Experimental Procedures. In parallel, a mock depletion was carried out on control beads in the absence of coupled p13 protein. lmmunoblotting for c&PA and cd&B of equivalent volumes of interphase cytosol after incubation with control beads (left panel) or ~13 beads (right panel) is shown. (8) Membrane release assays: 5 ul aliquots of interphase cytosol de pleted of cdcPA and cdc2-B were incubated with 30 ~1 aliquots of a binding reaction containing nuclear membrane vesicles bound to chromatin, in the absence (-okad. acid) or presence (+okad. acid) of 1 pM okadaic acid. Membrane binding was followed by fluorescence microscopy. DNA stain and membrane stain after 60 min incubation is shown.

nase/phosphatase regulatory system is contained in the membrane fraction. In a similar experiment, we tried to examine whether a regulatory target also resides on chromatin. To do this, frog sperm chromatin was incubated in interphase cytosol in the presence or absence of okadaic acid, as described above for the membrane fraction. After the 30 min incubation,

the samples

were

diluted

F&fold

with

buffer

to elimi-

nate the effect of interphase cytosol on membrane binding. This dilution step was necessary to “wash away” the cytosol from the sperm chromatin, because if decondensed chromatin is pelleted, it forms large aggregates that cannot be resuspended. When membranes were added to the cytosol-treated, dilution-washed chromatin, we observed that the membranes bound to the chromatin regardless of whether okadaic acid was present or absent during the initial cytosol incubation. Taken together with the result presented above, this observation strongly indicates that it is a membrane-localized component that is regulated by phosphorylation/dephosphorylation, rather than a component

bound

to chromatin.

In eukaryotic cells that undergo an open mitosis, the chromatin within the nucleus detaches from the nuclear envelope, the nuclear pores and the nuclear lamina disassemble, and the membrane component of the nuclear envelope vesicularizes at the beginning of mitosis. An un-

Cell 214

derstanding of the order of these events, their dependence on each other, and the mechanisms that regulate them is only now beginning to emerge. In an attempt to dissect the complex and manifold reactions involved in nuclear assembly into single steps, we have developed a simple in vitro assay to investigate the reversible binding of membrane vesicles to chromatin in the course of the cell cycle. This assay allows us to identify and characterize proteins that are involved in mediating and controlling the reversible binding of membranes to chromatin. Using inhibitors of phosphatases and kinases, we have demonstrated that membrane binding is dependent on the state of phosphorylation of a component that is most likely located on membranes derived from the nuclear envelope. Thus, when interphase extracts that normally promote membrane binding to chromatin were treated with the phosphatase inhibitor okadaic acid, these extracts now caused bound membranes to dissociate from chromatin. We have also shown that this okadaic acid-induced membrane release is dependent on ATP and sensitive to the kinase inhibitors 2-aminopurine and FSBA. These results strongly suggest that regulation of the observed membrane association with chromatin is determined by the equilibrium between the antagonistic activities of a kinaselphosphatase system. In light of the apparent participation of a phosphorylation regulatory system in membrane-chromatin association during the cell cycle, we investigated the role of MPF kinase. We found that membrane release is brought about by the activation of MPF in the cytosolic fraction. Furthermore, both by investigating the effect of pure MPF kinase on membrane-chromatin binding and by characterizing the release activity in c&?-depleted extracts, we have shown that MPF kinase does not cause membrane dissociation directly. Rather, our data indicate that MPF regulates the observed chromatin-membrane interaction indirectly, by modulating the activity of either kinases and/or phosphatases ultimately responsible for the phosphorylation state of their target(s) on the membrane. At present, we cannot say whether the enzymes assayed using inhibitors of kinases and phosphatases are the direct regulatory targets of MPF kinase. It is conceivable that the membranechromatin binding assayed in our system is subject to regulation by multiple enzymes, only a subset of which is under control of MPF kinase. Using our assay system to identify the enzymes involved, however, should allow us to clarify this question. In Figure 7, we present a simple model that fits our results most easily and outlines how a direct interaction of membranes and chromatin at the beginning and end of mitosis could be regulated. We propose that the binding of membrane vesicles to chromatin is mediated by a membrane-associated receptor (Wilson and Newport, 1988; Newport et al., 1990) that recognizes a ligand, most probably a protein, on the DNA. The binding equilibrium between chromatin and membrane vesicles is determined by the state of phosphorylation of the membrane receptor. When dephosphorylated, the receptor can bind to chromatin, and when phosphorylated, it cannot. The state of phosphorylation of the receptor is under the control of a phosphataselkinase regulatory system. When the equilibrium between these two antagonistic enzymes favors receptor

0 memb

Figure 7. Hypothesis on the Cell Cycle-Dependent Regulation Binding of Nuclear Membrane Vesicles to Chromatin

of the

Nuclear membrane vesicles contain a receptor that mediates binding lo a chromatin-associated ligand. The dynamic binding equilibrium is under the control of a phosphataselkinase regulatory system. Dephosphorylation, most probably of the membrane receptor, is prevalent in interphase, thus promoting binding. Phosphorylation is dominant during M-phase and causes release of chromatin-bound membranes. The shift in the binding equilibrium during mitosis is brought about by MPF, which alters the activities of the phosphataselkinase regulatory system involved. Notethat regulationof membrane binding may not necessarily occur by direct phosphorylation of the membrane receptor. As outlined in the text, it is also possible that phosphorylation of a cytosolic factor, which then interacts with the receptor, could regulate cell cycle-dependent receptor binding.

phosphorylation, vesicles dissociate from chromatin, and when dephosphorylation is favored, the vesicles bind chromatin. From our results, we believe that the regulatory phosphatase/kinase system that modulates vesicle-chromatin binding is controlled by the mitotic regulator MPF. As proposed in Figure 7, the activation of MPF at the beginning of mitosis causes membrane-chromatin dissociation either by increasing the activity of the kinase, which phosphorylates the receptor, or by decreasing the phosphatase activity, which dephosphorylates the receptor, or both. At the end of mitosis, the inactivation of MPF causes the kinaselphosphatase system to establish a new equilibrium, which favors dephosphorylation of the vesicle receptor. As a result of this dephosphorylation, vesicles rebind to chromatin in the first step of nuclear envelope assembly. We are aware of the fact that membrane release may not necessarily be caused by receptor phosphorylation. It is conceivable that at mitosis, a cytosolic factor in its phosphorylated form binds to the membranes and subsequently causes dissociation of membranes from DNA. Previously, we have shown that a trypsin-sensitive receptor is critical for the binding of membrane vesicles to chromatin during nuclear envelope assembly (Wilson and Newport, 1988). As outlined above, in our view the most likely way of regulating the observed membrane-chromatin binding is by phosphorylation and dephosphorylation of this membrane-bound receptor. In support of this, we have shown that when membrane vesicles are incubated in an interphase extract containing okadaic acid and then reisolated from this extract, they no longer bind chromatin. By contrast, vesicles incubated in the same extract lacking okadaic acid bind chromatin following reisolation. In an analogous experiment, no okadaic acid-sensitive dephosphorylation on chromatin has been observed. We believe that these results demonstrate several points. First, interphase extracts must contain both the phosphatase and

Assembly/Disassembly 215

of the Nuclear

Envelope

Membrane

kinase in an active form. If the phosphatase alone was active in interphase extracts, addition of okadaic acid would not have converted membranes to a nonbinding state. Second, by inhibiting the phosphatase with okadaic acid, the equlibrium between these two enzymes is shifted, so that phosphorylation is favored. Therefore, once fully phosphorylated, the reisolated membranes no longer bind chromatin. Third, the phosphorylated target resideson the membrane and not on chromatin. Finally, from the specificity of okadaic acid for phosphatases of the type 1 and 2A class, it appears quite likely that receptor phosphorylation occurs on serine and threonine sites. The identity of the regulated membrane receptor that binds chromatin has yet to be determined. The nuclear lamin proteins are a major component of the nucleus, located just below the inner nuclear envelope. In mammalian cells, it has been proposed that the membrane-bound lamin B acts to target membranes back to the chromatin surface at the end of mitosis (Burke and Gerace, 1988). At mitosis, lamin B appears to be directly phosphorylated by MPF kinase (Peter et al., 1990). In the case of lamins A and C, mitosis-specific phosphorylation sites on lamins have been identified (Ward and Kirschner, 1990) that are essential for lamin breakdown at mitosis (Heald and McKeon, 1990). However, no experimental evidence is available yet that would link lamin breakdown to the breakdown of nuclear envelope membranes at mitosis. In contrast to mammalian cells, only one lamin protein (called L,,,) has been found in Xenopus embryonic nuclei to date. However, a recent report by Doering and Stick (1990) indicates that an isoform to the only known lamin L,,, may exist. This isoform appears to be generated by alternative splicing and differs from Llll only in the C-terminal 12 amino acids. On an mRNA level, its abundance is far below that of LIII. While Llll is not membrane associated (Benavente et al., 1985; Newport et al., 1990) the L,,,isoform (which contains a C-terminal consensus sequence for isoprenylation; Doering and Stick, 1990) could, like mammalian lamin B, be membrane associated. The existence of this protein and its subcellular location, however, have yet to be demonstrated. Other proteins that might be good candidates for the currently unidentified membrane receptor would include the lamin B receptor (Worman et al., 1988) and several proteins identified as integral membrane proteins associated with the inner nuclear membrane (Senior and Gerace, 1988; Padan et al., 1990). Similarly, several proteins that localize to the nuclear periphery in interphase nuclei and to the surface of chromosomes at mitosis would begoodcandidatesfor thechromatin-bound protein that interacts with the receptor. These include perichromin (McKeon et al., 1984) peripherin (Chaley et al., 1984), and a recently identified 82 kd intranuclear scaffold protein isolated from rat liver nuclei (Fields and Shaper, 1988). The Role of MPF In Regulating MembraneGhromatln Association Our observation that membrane release can occur in interphase extracts when the phosphatase inhibitor okadaic acid is present demonstrates that the kinase that causes membrane release is present and active during inter-

phase. This observation by itself suggests that the regulatory kinase is not MPF for two reasons. First, the MPF kinase is only active at mitosis and not during interphase (Gerhart et al., 1984). Second, interphase extracts prepared in the presence of cycloheximide, as we prepared them, are unable to synthesize the cyclin subunits essential for activation of MPF. To definitively demonstrate that MPF is not directly responsible for membrane release, we have quantitatively depleted both Xenopus cdc2 homologs from an interphaseextract. We haveshown that these cdc2depleted extracts still cause release of membranes from chromatin when okadaic acid is present. In addition, we have shown that when pure MPF is added to a membrane-chromatin substrate, no membrane release is obsewed. Together, these observations strongly support our conclusion that membrane release, although regulated by MPF, is not caused directly by the MPF kinase. Our observations indicate that the kinaselphosphatase system that regulates envelope-chromatin association is present and active in interphase. During interphase, changes in nuclear size occur in response to DNA content and transcriptional activity, and during development. Therefore, the regulating system described here could be important in regulating envelope dynamics during interphase, as well as during mitosis. The indirect modulation of membrane release by MPF at mitosis is consistent with the original prediction that at mitosis, a component like MPF causes mitosis to occur by interacting with regulatory systems that control interphase cell dynamics (Warren, 1985). Thus, although MPF is known to regulate some mitotic events by directly phosphorylating the relevant substrates (e.g., histone Hl , lamins; Arion et al., 1988; Peter et al., 1990; see Moreno and Nurse, 1990, for a review), our results indicate that if a process is already subject to regulation during interphase by a preexisting system, then MPF may interact with and change this regulatory system, such that the substrates downstream are converted to a mitotic form. The regulation of membrane binding to chromatin is a clear example of this type of indirect regulation. Our results indicate that it is quite possible that many other cellular processes that are modified at mitosis and regulated during interphase will be subject to indirect regulation by MPF through their normal interphase regulatory systems. These include Golgi breakdown (Lucocq et al., 1987) changes in microtubule dynamics (Belmont et al., 1990), and exocytosis (Colman et al., 1985; Leaf et al., 1990; Kanki and Newport, submitted). Ex~rlmental

Procedures

Preparation

of Xenopus

Ego Extracts

X. laevis eggswereobtained, dejellied, packed, and lysed asdescribed previously (Newport, 19S7; Wilson and Newport, 19SS). Interphase extracts were prepared by using lysis buffer (250 mM sucrose, 50 mM KCI, 2.5 mM MgC&, 10 mM Hepes/NaOH [pH 7.41) supplemented with 1 mM dithiothreitol (OTT), 5 pg cytochalasin B per ml, aprotinin and leupeptin (10 pg/ml each), and 50 pg of cycloheximide per ml (to inhibit protein synthesis). Mitotic extracts were prepared by using EB buffer (So mM 5glycerophosphate [pH 7.21, 20 mM EGTA. 15 mM MgCl,, 1 mM DlT) supplemented with 0.5 mM TB-ATP (Newport and Spann, 1987; Dunphy and Newport, 19SS). Eggs were lysed by centrifugation for 10 min at 10,000 rpm in a Sotvall HB-4 rotor (Wilson and Newport, 19SS). The cytoplasmic layer (crude extract) was removed and further

Cdl 216

fractionated by centrifugation for 1 hr at 55,000 rpm in a Beckman TLS 55 rotor to yield cytosolic and membrane fraction. Interphase cytosol was supplemented with an ATP-regenerating system (Wilson and Newport, 1988) and spun for 20 min at 55,000 rpm to remove residual membranes, and aliquots, frozen in liquid nitrogen, were stored at -7OOC. Interphase membranes were mixed with 5-10 vol of lysis buffer supplemented with 1 mM ATP. 1 mM DTT, aprotinin (10 ullml), and leupeptin (10 ug/ml), and spun through a sucrose cushion of lysis buffer containing 0.5 M sucrose. Finally, the membranes were resuspended in cushion buffer (about IO-fold concentrated compared with the crude cytoplasmic fraction), and 5 ul aliquots were rapidly frozen in liquid nitrogen and stored at - 7OOC.

(UCSF). The recombinant cyclin 61 fusion protein was purified from an overexpressing E. coli strain by affinitychromatographyon glutathione Sepharose (Solomon et al., 1990) and the eluted cyclin construct was concentrated using a centricon 30 microconcentrator (Amicon). Purification of MPFwas as follows. The purified cyclin construct was incubated with interphase cytosol for 1 hr at 22OC. During that time period, MPF activity formed, as judged by measuring histone Hl kinase activity and/or the ability to induce nuclear breakdown in Xenopus interphase extracts (Dunphy and Newport, 1968). Formation of cdc2cyclin protein complex is a crucial step in this process. MPF was then purified by affinity chromatography on glutathione Sepharose. Elution of MPFoccurred with 5 mM glutathione, 10 mM Hepes/NaOH (pH 8.0).

Ammonlum Sulfate Precipitation of Interphase Cytosol Preparation of 30% AS-interphase was carried out as described previously for mitotic extracts (Wu and Gerhart, 1980; Dunphy and Newport, 1968). We diluted 1 vol of interphase cytosol with 1 vol of EB buffer. To 1 ml of the diluted cytosol, 0.43 ml of 3.6 M ammonium sulfate dissolved in EB was added, to yield a final concentration of 30% (v/v). The mixture was kept on ice for 30 min at O°C. Precipitated protein was isolated by centrifugation, dissolved in EB (4-fold concentrated in volume over the undiluted cytosol), and dialyzed against 100 vol of the same buffer. Aliquots were rapidly frozen in liquid nitrogen and stored at - 70°C.

Hlstone HI Klnsse Assay Histone Hl kinase activity of MPF was assayed exactly as described by Dunphy and Newport (1989). except a filter assay was employed to quantitate =P radioactivity incorporated into histone Hl. Specifically, kinase reactions were terminated after 10 min incubation at 22OC, by pipetting 15 ul aliquots (sample volume 20 ul) onto phosphocellulose filters. The filters were washed twice for 15 min in 1% phosphoric acid, once in 97% (v/v) ethanol (5 min), and finally air dried. Radioactivity was quantitated by liquid scintillation counting.

Binding of Nuclear Membranes to Frog Sperm Chromatin A standard binding reaction consisted of 180 ul EB or lysis buffer containing 0.5 mg polyglutamic acid per ml (Sigma; average molweight 51 kd), 2 frl of 0.2 M ATP (2 mM final concentration), 1 ul of 20 mM GTPTS (Boehringer Mannheims; 0.1 mM final concentration), 5 ul of membranes isolated from interphase extracts, and 5 ul demembranated Xenopus frog sperm chromatin (15,000 sperm per pl, isolated as described by Lohka and Masui, 1983 and Wilson and Newport, 1988). After 30 min incubation at 22’C, 30 ul aliquots of the binding reaction were employed in membrane release assays (described in detail in the figure legends). Binding of membranes to DNA was assessed visually by fluorescence microscopy (Wilson and Newport, 1988). employing the DNA dye Hoechst 33258 and the membrane dye DHCC. For each sample shown in the figures, at least 30 individual sperm were examined for membrane binding. After membrane release, DNA molecules could hardly be distinguished from the background in the membrane stain. Membrane release was judged to have occurred successfully only when virtually all DNA molecules examined had lost the distinctive membrane stain. Blndlng of Nuclear Membrane Vesicles to Chromstln after Prelncubatlon In Interphase Cytosol Approximately 25 pl interphase cytosol were incubated with 2.5 pl membranes, 0.1 mM GTP+, and 1 mM ATP. In a parallel sample, okadaic acid (1 urn final concentration) was included. After incubation for 30 min at 22OC. both samples were layered on 50 pl each of a cushion buffer containing 0.4 M sucrose in EB, 0.1 mM GTPyS and 1 mM ATP. Membranes were sedimented by centrifugation in an ep pendorf centrifuge (15 min at 4OC) and washed once with 0.3 ml of cushion buffer. The membrane pellets were resuspended in 5 ul of cushion buffer, and binding to demembranated frog sperm chromatin (see above) was assessed by fluorescence microscopy. Depletion of cdc2 by pl3-Afflnlty Chromatography Depletion of cdc2from interphasecytosol wascarried out asdescribed by Dunphy et al. (1988). Recombinant ~13 was isolated from an overexpressing E. coli strain as described (Brizuela et al., 1987) and coupled to CNBr-activated Sepharose as published (Dunphy et al., 1988). ~13 beads were incubated with interphase cytosol by gentle rotation for 1 hr at 4OC and spun down, and the supernatant, rapidly frozen in liquid nitrogen, was stored at - 70°C. The depleted cytosol was checked for residual cc/c2 by Western blotting. Afflnlty Purlflcation of MPF Purification of a recombinant cyclin El construct was as follows. A truncated cyclin Bl cDNA clone from sea urchin (missing the first 13 N-terminal amino acids) and fused to a portion of glutathione S-transferase was generously provided by Mark Solomon and Marc Kirschner

Electrophoretlc Techniques Proteins were resolved on 10% SDS-polyacrylamide gels (Laemmli. 1970) transferred to nitrocellulose (Finlay et al., 1987) probed with anti-cdc2 antibodies and ‘251-protein A, and analyzed by fluorography as described by Dunphy and Newport (1989). Antlbodlss Antibodies against cdcPA homolog overexpressed in pressed protein. Antibodies oghue) were obtained by peptide of the protein from Materials Okadaic

acid was obtained

were raised in rabbits against the yeast E. coli and affinity purified from the overexagainst cdcbB (kindly provided by D. Donimmunization with a synthetic C-terminal X. laevis.

from Moana

Bioproducts,

Hawaii.

Acknowledgments We would like to thank Mark Solomon and Marc Kirschner for providing the cyclin fusion construct, as well as for their generous advice on how best to prepare the fusion protein. We would also like to thank Dan Donoghuefor providing anticdc2-B antibody for use in this work, Philip Cohen for providing phosphatase inhibitor peptide, and Shirley Allen for help in preparing the manuscript. This work was supported by a fellowship from the Deutsche Forschungsgemeinschaff to Ft. P. and by National Institutes of Health grant GM 33523-07 to J. N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement in accordance with USC 18 Section 1734 solely to indicate this fact. Received

December

4, 1990; revised

January

30, 1991

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Moreno, veritas?

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in vivo drives

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assemfunc-

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M phase

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Lohka, M., and Masui. Y. (1983). Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmlc compo nents. Science 220, 719-721. Lucocq, J., Pryde, J., Berger, E., and Warren, 0. (1987) A mitotic form of the Golgi apparatus in HeLa cells. J. Cell Biol. 104.865-674. McKeon, F. D., Tuffanelli, D. L., Kobayashi. S.. and Kirschner, M. W. (1984). The redistribution of a conserved nuclear envelope protein during the cell cycle suggests a pathway for chromosome condensation. Cell 36, 63-92. Make-Lye, R., and Kirschner, events in a cell-free system.

M. W. (1985). Induction Cell 41, 165-175.

of early mitotic

Note

Added

In Proof

The authors of the manuscript submitted” are W. 0. Dunphy

referred to throughout and J. Newport.

as “Newport,