The nuclear envelope lamina is reversibly depolymerized during mitosis

Cell, Vol. 19. 277-287.

January

1980,

Copyright

0 1980 by MIT

The Nuclear Envelope Lamina Is Reversibly Depolymerized during Mitosis

Larry Gerace and Gunter Blobel Laboratory of Cell Biology The Rockefeller University New York, New York 10021

Summary The nuclear envelope lamina is a supramolecular protein assembly associated with the nucleoplasmic surface of the inner nuclear membrane, which contains three predominant polypeptide components in mammalian cells (lamins A, B and C). We previously demonstrated by immunofluorescence microscopy that the lamina is reversibly disassembled during cell division, coincident with the disassembly and reconstruction of the mitotic nuclear envelope architecture. In this paper, these immunocytochemical observations are extended with cell fractionation and immunoprecipitation studies performed on synchronized populations of tissue culture cells. With these techniques, we have established that during mitosis, lamins A and C occur in a soluble and nonmembrane-associated state. In contrast, the mitotic lamin B may be associated with membrane fragments derived from the disassembled interphase nuclear envelope. From sedimentation analysis on sucrose gradients, we have determined that all three lamins are monomeric at periods of mitotic lamina disassembly. These results, together with quantitative immunoprecipitation studies, demonstrate that the lamina is reversibly depolymerized during cell division. Attendant with the depolymerized state of the lamina, the mitotic lamins (which are phosphoproteins) have a distinctly more acidic isoelectric point and a substantially higher level of phosphorylation compared to their interphase counterparts. This indicates that reversible enzymatic phosphorylations of the lamins may be involved in modulating the state of polymerization of the lamina and its reversible mitotic disassembly. Introduction Recent investigations have suggested that an important element in the structure and dynamic behavior of the eucaryotic nuclear envelope (for nuclear envelope reviews see Kay and Johnston, 1973; Franke. 1974; Fry, 1976) is the nuclear envelope lamina, a supramolecular assembly of peripheral membrane proteins which is associated with the nucleoplasmic surface of the inner nuclear membrane (Dwyer and Blobel, 1976; Gerace, Blum and Blobel, 1978). The lamina has previously been isolated from rat liver nuclear envelopes, occurring together with morphologically associated nuclear pore complexes as a stable supramo-

lecular structure (the “pore complex-lamina fraction”; Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). lmmunocytochemical studies have established that the rat liver lamina contains predominantly three polypeptide components (Gerace et al., 1978). With anti-* bodies to these proteins, it was demonstrated by immunofluorescence microscopy that the lamina is completely and reversibly disassembled during mitosis, coincident with the disassembly and reconstruction of the nuclear envelope (Gerace et al., 1978; Krone et al., 1978). In this paper, we have further investigated the mitotic behavior of the lamina with cell fractionation and immunoprecipitation experiments performed on synchronized populations of tissue culture cells. These studies demonstrate that the lamina undergoes reversible depolymerization during cell division, a process that may result from reversible enzymatic phosphorylation. We discuss the role of this reversible lamina depolymerization in the structural dynamics of the nuclear envelope during mitosis. lmmunoprecipation of the Lamina Polypeptides from CHO Cells The three major polypeptide components of the rat liver lamina migrate at 70, 67 and 60 kilodaltons on SDS gels (Gerace et al., 1978). lmmunofluorescence microscopy (Gerace et al., 1978; Krone et al., 19781, in combination with immunoprecipitation and immunoautoradiographic analyses (L. Gerace, A. Blum and G. Blobel, manuscript in preparation), indicate that the presence of these three distinct molecular species is a general feature of the lamina of numerous types of higher eucaryotic cells. We suggest the nomenclature lamin A, lamin B and lamin C for the 70, 67 and 60 kilodalton lamina proteins, respectively. For the experiments described in this study, we have used the Chinese hamster ovary (CHO) cell line. Antibodies raised against lamin A from rat liver were used for immunoprecipitation of the CHO lamins. As a first step in our immunoprecipitation procedure, proteins were completely solubilized by boiling in SDS to disrupt noncovalent protein-protein interactions. With rat liver nuclear envelopes prepared under nonoxidizing conditions, this solubilization procedure renders the three lamins completely monomeric, as analyzed by nonreducing SDS gel electrophoresis (data not shown). Our preparation of anti-lamin A antibodies specifically immunoprecipitates three polypeptides from unfractionated CHO cells (Figure 1 A, lane “cells”), that co-migrate with the three rat liver lamins (Figure 1 A, lane “NE”) on SDS gels. Based on two-dimensional gel electrophoresis (see Figure 4) and peptide mapping studies (L. Gerace, A. Blum and G. Blobel, manuscript in preparation), each of these immunoprecipitated CHO polypeptides biochemically corresponds to

Cell 270

B.

C.

INTER PHASE NE

Figure

I.

Fractionation

cells

(NI)

cells

and lmmunoprecipitation

-TRlTON P140 s140

+TRITON P140 s140

MITOSIS cells

-TRITON D140 ~140

+TRITON D140 ~140

of CHO Cell Populations

?S-methionine-labeled interphase (B) or mitotic (C) populations were homogenized as described in Experimental Procedures and were divided into two aliquots. Triton X-i 00 was added to a 2% final concentration to one of these samples (+Triton). and both were then centrifuged, yielding a pellet (~140) and a post-microsomal supernatant (~140). These samples, together with aliquots of labeled unfractionated interphase (A, cells) or mitotic (C. cells) populations, were immunoprecipitated with anti-lamin A antibodies as described in Experimental Procedures. In addition, a sample of unfractionated interphase cells was processed for immunoprecipitation with nonimmune chicken IgG [A, (NIL cells] to define the nonspecific background bands in the other immunoprecipitates. Following SDS gel electrophoresis of these samples, fluorography was performed. A composite from different gels is shown. For a standard, an electropherogram of rat liver nuclear envelopes (NE), stained with Coomassie Blue, is included. Histones. which are present in nuclear envelopes prepared in this fashion (Dwyer and Blobel, 1976), are indicated by dots to the lefl of this lane. In the experiment of (B). the lamin A band shows a diffuse leading edge, which we attribute to slight proteolytic degradation of this protein.

the co-migrating rat liver protein. Approximately 0.030.05% of the total 35S cpm from samples of exponentially growing CHO cells is immunoprecipitated in each of the three lamin bands (see Table 1). Since lamins A and C from rat liver are both immunologically and structurally similar, as determined by Ouchterlony double diffusion (Gerace et al., 1978) and peptide mapping procedures (L. Gerace, A. Blum

and G. Blobel, manuscript in preparation), one would expect both of the homologous CHO polypeptides to be immunoprecipitated by anti-lamin A antibodies. Lamin B. in contrast, has a distinct cyanogen bromide peptide map from the other two lamins (L. Gerace, A. Blum and G. Blobel, manuscript in preparation). Nevertheless, this protein is also immunoprecipitated by our preparation of anti-lamin A antibodies. This

Nuclear 279

Table

Envelope

1. Stability

Lamina

during

of the Lamins

Mitosis

through

lmmunoprecipitated

Mitosis

cpm

Gl cpm

Interphase

Mitosis

Gl

Mitosis cm

Lamin A

2263

1430

1243

0.87

Lamin

B

2360

1659

1628

0.98

Lamin

C

1547

1048

1087

1.03

Cells were grown for 2.2 hr in Ham’s FlO medium (see Experimental Procedures) containing 5 pCi/ml 35S-methionine. Following 1 additional hr of growth in nonradioactive medium, mitotic cells were obtained by pooling eight sequential shake-offs. One half of the collected mitotic cells was frozen. The other half was returned to 37°C culture for 1 hr, at which time >95% of the cells in this sample were in early Gl Samples of mitotic and early Gl cells, and exponentially growing interphase cells (from the same culture) each containing 5250,000 initial 35S cpm. were processed for immunoprecipitation with anti-lamin A antibodies. Following electrophoresis of these samples on an SDS gel and visualization of the lamin bands by fluorography. bands were excised and radioactivity was determined (see Experimental Procedures). Approximately 1.5 times the cpm are immunoprecipitated in the lamin bands from exponentially growing interphase cells, as from synchronized mitotic or Gl populations. A similar result was obtained with cultures that were labeled continuously for 45 hr (data not shown). These findings can be explained by the possibility that in an exponentially growing interphase population, the lamins on average comprise a greater percentage of the total cellular protein than in a mitotic cell population.

result may indicate that lamin B contains one or several domains that are immunologically similar to portions of lamin A. Alternatively, this result can be explained by the possibility that the lamin A preparation which we used for immunization contained immunogenic quantities of lamin B as well, despite the fact that our lamin A antigen appeared homogenous by SDS gel electrophoresis (Gerace et al., 1978). Depolymerization of the Lamina during Mitosis Results from immunofluorescence microscopy (Gerace et al., 1978; Krone et al., 1978) suggest that during mitosis the lamina is completely disassembled at the resolution of light microscopy and that the proposed interphase association of the lamins with chromatin (Gerace et al., 1978) is abrogated. These results, however, do not indicate whether the lamins (which form an insoluble supramolecular assembly in interphase) are depolymerized to a monomeric state during cell division, or whether they are disaggregated to form regular or irregular oligomers during this period. Moreover, these studies do not determine whether the lamins (peripheral membrane proteins in interphase) lose their association with membranes when the nuclear envelope is disassembled to form small vesicles during mitosis (morphologically described by Robbins and Gonatas, 1964; Murray, Murray and Pizzo, 1965; Erlandson and DeHarven, 1971). We have addressed these questions with studies involving fractionation of mitotic CHO cells. Synchronized mitotic cell populations were ob-

tained by mechanical shake-off of monolayer cultures (Tobey, Anderson and Peterson, 1967). With our synchrony conditions, >90% of these mitotic cells represented metaphase and early-mid anaphase stages. The lamina is completely disassembled at these periods of CHO cell division, as determined by immunofluorescence examination of synchronized mitotic populations (data not shown). lmmunoprecipitation of (unfractionated) mitotic cells with anti-lamin A antibodies (Figure lC, lane “cells”) demonstrates that the three lamins occur in an apparently undegraded state when the lamin is disassembled; no additional antigenie species are detectable. To accomplish cell fractionation, mitotic or (control) interphase cell populations were homogenized under various conditions, and homogenates were subjected to high speed centrifugation, yielding a ~140 (insoluble or pellet fraction), and an ~140 (soluble or postmicrosomal supernatant fraction). Subsequent immunoprecipitation of these fractions with anti-lamin A antibodies permitted a determination of the subcellular distribution of the lamins in a balance sheet fashion (Figure 1). If these proteins are membrane-associated or if they occur in the form of moderately large aggregates at periods of mitotic lamina disassembly, they would appear in a ~140 fraction. Alternatively, the proteins would occur in an ~140 fraction only if they are soluble and not membrane-associated. When interphase cell homogenates are fractionated under isotonic conditions, the lamins occur exclusively in a ~140 fraction (Figure 1 B, -Triton; compare lanes “~140” and “~140”). This result is anticipated, since the rat liver lamina is structurally stable throughout a wide range of ionic strength (Dwyer and Blobel, 19761, including the conditions of this experiment. In contrast, when mitotic cell homogenates are fractionated in a similar fashion, more than 50% of lamins A and C is found in the ~140 fraction (Figure 1 C, -Triton; compare lanes “~140” and “~140”). Hence under these experimental conditions at least a substantial portion of these two proteins is soluble and not membrane-associated when the lamina is in a disassembled state. Lamin B, on the other hand, appears mostly in the ~140 fraction of mitotic homogenates (Figure 1 C; compare lanes “~140” and “~140”). To investigate whether the apparently insoluble state of this protein during mitosis is due to an association with membrane structures, homogenates were treated with Triton X100 to solubilize membranes before fractionation. This treatment does not modify the fractionation pattern in control interphase cell homogenates (Figure 1 B, +Triton; compare lanes “~140” and “sl40”), since the interphase lamina is a supramolecular assembly that is not dependent on the presence of nuclear envelope lipids for its integrity (Dwyer and Blobel, 1976). As a consequence of Triton treatment of mitotic cell homogenates, however, lamin B is rendered almost com-

Cflll 200

pletely soluble (Figure lC, +Triton; compare lanes “~140” and “~140”). This result suggests that during mitosis lamin B may be predominantly associated with membranes. Since Triton treatment does not increase the relative soluble proportion of lamins A and C in mitotic cell homogenates (Figure 1C; compare lanes -Triton “~140” and +Triton “sl40”), the “insoluble” mitotic lamins A and C are apparently not membrane-associated. We considered it probable that the partial presence of these two proteins in the mitotic ~140 fraction was a consequence of nonspecific adsorption of soluble lamins A and C to the surfaces of cellular debris and particulate matter, occuring during the processes of cell disruption and/or subsequent manipulations. Such a redistribution of soluble proteins commonly occurs during cell fractionation procedures (see, for example, Scheele, Palade and Tartakoff, 1978). After extensive investigation of this phenomenon (see Experimental Procedures), it became apparent that mild cell homogenization conditions and/or elevated ionic strength in the fractionation medium favored an increase in the relative soluble proportion of lamins A and C in mitotic homogenates. If instead of using a mechanically vigorous cell disruption approach we accomplish lysis of mitotic cells by gentle vortexing in isotonic buffer containing Triton X-l 00, there is a nearly complete appearance of lamins A and C (as well as the possibly membraneassociated lamin B) in the ~140 fraction (Figure 2, M; compare lanes “~140” and “~140”). This suggests that lamins A and C, rather than being only partially soluble during mitosis, occur nearly quantitatively in a soluble form. To determine the sedimentation characteristics of the lamins during mitosis, an ~140 fraction obtained by detergent lysis of mitotic cells was centrifuged on a linear sucrose gradient, and gradient fractions were immunoprecipitated with anti-lamin A antibodies (Figure 3). From this analysis it is evident that the three lamins (which migrate at 60-70 kilodaltons on SDS gels) sediment exclusively at the position of bovine serum albumin (molecular weight 66 kilodaltons). Since the ~140 fraction examined in Figure 3 contains nearly the entire mass of the lamins present in mitotic cells (see Figure 21, this result indicates that the lamina is completely depolymerized to a monomeric state during mitosis. When a mitotic ~140 fraction obtained by nondetergent cell lysis (Figure 1) which contains primarily lamins A and C is similarly analyzed, these two proteins sediment exclusively as monomeric species (data not shown). This demonstrates that the monomeric state of lamins A and C is not induced by detergent treatment. Reversible Nature of Lamina Depolymerization lmmunofluorescence microscopy strongly suggests that the mitotic lamina depolymerization is a reversible

I

M

cells p140 s14Q P140 a40

Figure

2. Fractionation

of CHO Cells following

Detergent

Lysis

Pellets of 35S-methionine-labeled interphase (I) or synchronized mitotic (M) cells were resuspended in isotonic buffer containing 1% Triton X-i 00 and were vortexed gently to accomplish cell disruption (see Experimental Procedures). These samples were then centrifuged, yielding a pellet (~140) and a post-microsomal supernatant (~140). These fractions, together with an aliquot of unfractionated interphase cells (I cells) as a standard, were immunoprecipitated with anti-lamin A antibodies. A fluorograph of an SDS gel of these immunoprecipitates is shown. The lamins of the interphase cell population are solubilized (that is, are present in an ~140 fraction) to a negligible degree by this detergent lysis. For fractionated mitotic cells, 80% of the total immunoprecipitated CPM of the three lamins is present in the ~140 fraction and 20% in the pi40 fraction, as determined by excision of gel bands and scintillation counting (see Experimental Procedures). Since the mitotic index in this experiment was 90%. only -90% of the total lamin mass in this experiment would be theoretically soluble.

process, with the disassembled lamins being recycled for the formation of the new telophase nuclear envelopes. An alternate hypothesis, that the lamina is disassembled by proteolytic degradation of its con-

Nuclear Envelope Lamina during Mitosis 281

ggal (169 1

cat (11.39 I

w (7.29 I

BSA (4.5s) I

Figure 3. Sucrose Gradient Sedimentation of Mitotic Cell ~140 Fraction Obtained by Detergent Lysis An ~140 fraction obtained from mitotic cells (see Figure 2) was layered on a 5-20% sucrose gradient (5 ml) made with Triton lysis buffer and was centrifuged for 7.5 hr at 48,000 rpm in a Beckman SW50.1 rotor. Fractions were collected, and, after TCA precipitation in the presence of 50 r.rg of carrier tRNA. were immunoprecipitated by anti-lamin A antibodies. Material occuring in the pellet region a

I

of the gradient (Pel), together with unfractionated interphase cells (I cells) were similarly processed. A fluorograph of an SDS gel of these samples is shown. Direction of sucrose gradient sedimentation (arrow) is from right to left. The positions of protein standards sediments on parallel gradients are indicated: bovine serum albumin (BSA). human IgG (IgG), bovine catalase (cat) and E. coli /3-galactosidase (p gal).

stituent polypeptides during prophase and reassembled only from proteins synthesized de novo during mitosis, has no support from our data. Similar quantities of each of the three radioactively labeled lamins can be immunoprecipitated from mitotic cells (in which the lamina is disassembled), as from comparable masses of interphase cells (Table 1). Since the radioactively labeled proteins analyzed in our experiments represent only polypeptides synthesized prior to mitosis (Table 1 and Figures l-3), these results argue against a putative degradation of the lamins during prophase. Furthermore, similar quantities of these three polypeptides can be immunoprecipitated from early Gl cells, as from mitotic cells (Table 1). In these Gl populations, the lamin antigens occur exclusively in a perinuclear localization reflective of the polymerized lamina. This implies that the depolymerized lamins are recycled at the conclusion of mitosis for the formation of the lamina in the progeny cell nuclear envelopes, and therefore that the mitotic depolymerization of the lamina is a reversible process. A Possible Mechanism of Lamina Depolymerization We anticipated that the process of reversible lamina depolymerization may be accomplished by reversible post-synthetic modifications involving the three lamins. Since many post-translational modifications induce a change in protein isoelectric point (Uy and Wold, 1977), we compared the isoelectric properties of the lamins from mitotic cells and exponentially growing interphase cells by two-dimensional gel elec-

trophoresis (Figure 4). In these experiments, electrophoresis of the first dimension separates most proteins according to their isoelectric points, even though more basic proteins are not at isoelectric equilibrium (O’Farrell. Goodman and O’Farrell, 1977). For the interphase cell immunoprecipitate, lamins A and C have a closely similar migration in the pH gradient of the first dimension (Figure 4, panel I). Also, they occur as apparently multiple isoelectric forms, represented as a family of approximately equally separated spots. Lamin B has a significantly lesser migration in the first dimension than the other two lamins, and is isoelectrically more homogenous. At isoelectric equilibrium, lamins A and C have a pl of -7.5; the pl of lamin B is -6.0 (data not shown). The lamins from the immunoprecipitate of mitotic cells also show a complex migration pattern of multiple spots in the first dimension, although individual spots are poorly defined in this case (Figure 4, panel MI. Nevertheless, it is apparent that for each of the three mitotic proteins the average migration of the composite protein pattern is distinctly diminished toward the basic end of the pH gradient, compared to that of the corresponding interphase proteins. This indicates that the average isoelectric point of each of the three lamins is more acidic during mitosis, suggesting that all three proteins are subjected to post-synthetic modifications during this period. Enzymatic phosphorylation is a common post-synthetic protein modification that would impart a more acidic isoelectric point on proteins. Also, it is known to exert important regulatory effects in a number of

Cell 282

r

Basic

Acidic

NEPHGE

biological systems (Rubin and Rosen, 1975). We therefore examined pulse-labeled CHO cells for the incorporation of 32P into the lamins from both mitotic and interphase cells. As shown in Figure 5, these proteins incorporate 32P both during interphase and immediately prior to, or during, mitosis. At least most of this incorporated 32P has the enzymatic and chemical characteristics (see Bitte and Kabat, 1974) of a protein-associated phosphomonoester linkage-that is, phosphoserine and phosphothreonine (data not shown). For both pulse-labeled mitotic and interphase cells, the 32P incorporated into anti-lamin A immunoprecipitates (see Figure 5) is sensitive to digestion with proteinase K but insensitive to treatment with DNAase and RNAase. It is not extracted from an aqueous phase by chloroform:methanol (2:l). Furthermore, the incorporated label is stable to treatment with 1 M hydroxylamine, 1 tvl succinic acid (pH 5) (1 hr at 37’C); and to 10% TCA (>75% stable in 20 min at 90X), but is removed by treatment with 1 N NaOH (~85% labile in IO min at 9O’C). Finally, most of the 32P label incorporated in each of the three lamins is removed by treatment with E. coli alkaline phosphatase. Enzymatic phosphorylation of the lamins during interphase growth could clearly account for the appar-

Figure 4. Two-Dimensional Gel Electropholesis of the Lamins lmmunoprecipitated from Interphase or Mitotic Cells Samples of 35S-methionine-labeled exponentially growing interphase (I) or synchronized mitotic (M) cells were solubilized in SDS, immunoprecipitated with anti-lamin A antibodies and resolved by nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension (O’Farrell et al., 1977). followed by SDSpolyacrylamide gel electrophoresis (SDSPAGE) in the second dimension (see Experimental Procedures). Shown are portions of these two electropherograms, visualized by fluorography. that have been precisely aligned with respect to the origin of the first-dimensional electrophoresis. Several background nonspecific spots that are invariant in firstdimensional migration between interphase and mitotic cells have been connected with solid lines to provide an additional frame of reference. The dotted lines indicate the approximate average change in first-dimensional migration for the three lamin proteins between interphase and mitosis.

ently multiple isoelectric forms of the interphase proteins seen by two-dimensional gel electrophoresis (Figure 4). If phosphorylation were also responsible for the acidic isoelectric shift of the lamins in mitotic cells (Figure 41, then the average level of phosphorylation of these proteins should be higher in mitosis than in interphase. To examine this question, CHO cells were subjected to continuous, long-term radioactive labeling (36 hr) in medium containing 3H-leutine and 32P-orthophosphoric acid. The ratio of 32P/3H radioactivity in each of the three lamins was determined for proteins immunoprecipitated from interphase, mitotic and early Gl cell populations. We assumed that the long term conditions of labeling in this experiment provide a roughly steadystate situation for incorporation of these labels. The 32P/3H ratio for a particular lamin at any one of the three cell cycle stages would therefore be directly proportional to the level of phosphorylation of the polypeptide at that particular stage. This permits a direct comparison of relative phosphorylation between the different cell cycle populations. As expected, the average level of phosphorylation of all three lamins is increased in mitotic cells compared to exponentially growing interphase populations (Table 2). This increase is dramatic, amounting to a

Nuclear 283

Envelope

Lamina

during

Mitosis

which the lamins occur exclusively in a PerinUClear disposition, the average level of phosphorylation is intermediate between that of mitotic and exponentially growing interphase populations. Discussion

Figure 5. SDS Gel Electrophoresis Labeled Interphase or Mitotic Cells

of the Lamins

from

32P Pulse-

Samples of 35S-methionine-labeled interphase (I) cells or 32P pulselabeled exponentially growing interphase or synchronized mitotic (M) cells were immunoprecipitated with anti-lamin A antibodies and electrophoresed on an SDS gel. Visualization was by autoradiography. A DuPont Hi-Plus intensifying screen was used to enhance ‘*P lanes.

more than 3 fold augmentation for lamins A and C and a more than 6 fold increase for lamin B. For populations of cells that have proceeded into early Gl, in

Previously, studies by immunofluorescence microscopy have documented the changes occuring during mitosis in the cellular localization of the three predominant lamina polypeptides (Gerace et al., 1978; Krone et al., 1978). On the basis of our earlier investigations, we concluded that the lamina is completely disassembled during cell division at the resolution of light microscopy, in an apparently reversible fashion. In this paper, we have used cell fractionation and immunoprecipitation procedures to investigate several molecular aspects of this process. Specifically, we considered it important to determine whether the lamins, which constitute a presumptive polymeric assembly during interphase, are depolymerized during mitosis to a monomolecular state. Furthermore, since the interphase lamins are peripheral membrane proteins associated with the inner nuclear membrane, we wished to determine whether they remained associated with membrane fragments derived from the disassembled nuclear envelope during cell division. From our experiments, we conclude that the lamina is completely depolymerized during mitosis to a monomeric form of the three lamins. Furthermore, lamins A and C are soluble and are not membrane-associated during this period. In contrast, most of lamin B appears to be associated with membrane structures in mitotic cell homogenates. This may indicate that lamin B retains a physiologically meaningful interaction with membrane fragments of the disassembled nuclear envelope subsequent to lamina depolymerization. The immunoprecipitation results presented in this study effectively exclude the possibility that the lamina is disassembled in prophase by proteolytic degradation of the lamins (Table 1, Figures 1 and 2). Rather, our results indicate that after the lamina is disassembled, the lamins occur in a an apparently intact form. Furthermore, these depolymerized proteins are at least predominantly conserved and recycled in the formation of the daughter cell nuclear envelopes (Table 1). The important conclusion that can be made from these results, therefore, is that the mitotic lamina depolymerization is a reversible process. We have ascribed two discrete biochemical differences to the depolymerized lamins in this study. On average, during cell division these proteins have both a more acidic isoelectric point and a substantially higher level of associated phosphorus compared to their interphase counterparts. Considering the large body of evidence that now exists implicating reversible enzymatic phosphorylation as a physiological modulator of protein function (Rubin and Rosen, 19754, it

Cell 284

Table

2. Determination

of the Relative

Levels

of Phosphorylation

of the Lamins

in Different

Cell Cycle

Populations

Interphase

Mitosis

Gl

lmmunoprecipitated cpma

lmmunoprecipitated cpm’

lmmunoprecipitated cpma

3H

32P

32P/3H

%I

=P

32P/3H

3H

32P

32P/3H

Lamin A

542

110

0.20

322

215

0.67

245

92

0.38

Lamin 6

418

48

0.12

262

189

0.72

196

55

0.28

Lamin C

350

83

0.24

207

130

0.63

177

61

0.35

Cells were maintained in Ham’s F10 medium (phosphate-free) containing 10% nondialyzed fetal calf serum with 1 .O pCi/ml 3H-leucine and 10 &i/ml “P-phosphate for 36 hr of exponential growth. Then mitotic cells were,collected by pooling five sequential shake-offs, being maintained in radioactive growth medium at all points during mitotic selection. One half of the pooled mitotic cells was frozen. The other half was returned to 37°C culture in radioactive growth medium for 50 min. at which time >90% of the cells were in early Gl. Samples of mitotic. early Gl or exponentially growing interphase cells (from the same culture used for mitotic selection) were immunoprecipitated with anti-lamin A antibodies and electrophoresed on an SDS gel. Following visualization by autoradiography, the lamin bands were excised and radioactivity was determined (see Experimental Procedures). Samples were analyzed in a Beckman LS 8000 liquid scintillation counter using an AQC program that gave negligible spillover between ‘H and 32P channels. a Samples were corrected for a background of 20 cpm. 3H; and 20 cpm. 32P,

seems probable that phosphorylation is involved in the reversible depolymerization of the lamina during cell division. However, since it is also possible that additional post-synthetic enzymatic modifications participate in lamina dynamics, this problem must be subjected to greater scrutiny before final conclusions can be made on the mechanism of lamina depolymerization. We note that a nuclear envelope-associated protein kinase activity has been described (Lam and Kasper, 1979). The mitotic behavior and polymeric characteristics of the lamins described in the present study support the possibility that the lamina is directly related to the structural dynamics of the nuclear envelope during the cell cycle (Gerace et al., 1978). Basically, this type of principle (that a supramolecular assembly of peripheral membrane proteins is involved in determining more general structural properties of the corresponding membrane) is known to be relevant to the organization of a number of other biological membranes, notably the red cell plasma membrane (see, for example, Steck, 1974; Marchesi, Furthmayr and Tomita, 1976; Sheetz and Singer, 1977; Shotten et al., 1978). Considering the data in this paper, the prophase depolymerization of the lamina, combined with a dissociation of the lamin interactions with chromatin and (for lamins A and C) the inner nuclear membrane, may express necessary conditions of the disassembly of the nuclear envelope. Conversely, the repolymerization of the lamina in telophase, occurring together with a reassertion of its specific interactions with chromatin and with nascent nuclear membranes, may catalyze important steps in the reconstruction of the nuclear envelopes of progeny cells. This proposed involvement of the lamina in the reconstruction of the mitotic nuclear envelope is conceptually similar to hypotheses concerning the matu-

ration of enveloped animal viruses at the host cell surface [for example, influenza virus (Lenard and Compans, 1974) and Semliki Forest virus (Garoff and Simons, 197411. In the case of influenza virus, for example, it has been proposed (Lenard and Compans, 1974) that the viral matrix protein (a membrane-associated protein which may be functionally analogous to the lamins), by means of an interaction with both the viral nucleoprotein complex and with the host cell plasma membrane, mediates a physical association between these two components that ultimately results in lipid envelopment of the viral nucleocapsid. The three lamins are believed to constitute a polymeric structure, based on a number of general biochemical and ultrastructural criteria (Dwyer and Blobel, 1976; Gerace et al., 1978; this study). The precise molecular arrangement of these three polypeptides in the lamina, however, remains to be determined. We wish to point out that the rat liver lamina is structurally stable when exposed to most sequences of extraction conditions currently used by various groups to subfractionate nuclei to obtain basic nuclear architectural elements [for example, nuclear matrix (Comings and Okada, 1976; Berezney and Coffey, 19771, nuclear skeleton (Miller, Huang and Pogo, 19781, hnRNP fibers (Herman, Weymouth and Penman, 1978) and so on]. It is therefore anticipated that the lamina would be present in these preparations, either more or less ultrastructurally well preserved. For example, the three lamins are apparently the predominant polypeptides in “nuclear protein matrix” material fromrodent liver (Comings and Okada, 1976; Berezney and Coffey, 1978). We emphasize, however, that the lamins, by a number of different immunocytochemical and immunological approaches (Gerace et al., 1978; Krone et al., 19781, localize exclu-

Nuclear 285

Envelope

Lamina

during

Mitosis

sively at the periphery of the nucleus, and are not constituents of the “protein matrix” that extends through portions of the nuclear interior. Experimental

Procedures

Cell Culture and Synchrony Chinese hamster ovary cells, obtained from R. Tobey (University of California at Los Alamos. New Mexico), were grown in monolayer culture in modified (Tobey et al., 1967) Ham’s FlO medium containing 10% fetal calf serum (GIBCO), 0.02 M HEPES buffer, 75 U/ml penicillin and 75 pg/ml streptomycin. For most mitotic synchrony experiments, cells were initially plated in 150 cm2 plastic tissue culture flasks (Corning) at 4 x 1 O6 cells per flask. Following 24-30 hr of growth, cultures were radioactively labeled for 8-12 hr with complete growth medium containing 10 @Ci/ml of +-methionine (Amersham). Subsequently, radioactive medium was removed and mitotic cells were obtained from labeled cultures by mechanical shake-off (Tobey et al., 1967). Mitotic shake-offs were conducted at room temperature after 15 min intervals of cell growth at 37°C. Following each shake-off, medium containing mitotic cells was immediately chilled to 0°C by gentle swirling in an ice bath. Cells from the first several shake-offs were discarded. The next successive 4-8 shakes were pooled and used for subsequent experiments. Under these conditions of harvesting, 190% of the pooled cells were in metaphase and early-mid anaphase stages of cell division. For some experiments, early Gl cell populations were obtained by warming synchronized mitotic populations to 37’C and returning these cells to an incubator for 45-60 min. Exponentially growing interphase populations were obtained by chilling tissue culture flasks on ice and then scraping cells into cold phosphate-buffered saline (PBS) with a rubber policeman. A single-step presynchronization of cultures with thymidine was used to increase mitotic cell yields in experiments involving pulse labeling with ‘*P. For this procedure, exponentially growing cultures were maintained in Ham’s FlO medium containing 0.002 M thymidine (Bostock, Prescott and Kirkpatrick, 1971) for 9 hr. Thymidine medium was then removed and flasks were returned to culture with normal growth medium for an additional -5 hr (until the beginning of a wave of mitosis). The medium was decanted and flasks were rinsed well with phosphate-free saline buffered with 0.02 M HEPES (pH 7.4). Next, prewarmed phosphate-free growth medium containing 32Porthophosphoric acid (New England Nuclear) at a concentration of 50-150 pCi/ml was added to flasks, which were returned to 37°C for 20 min. After this period, four successive mitotic cell shake-offs were performed as described above. The second to fourth shakes were pooled for pulse-labeled mitotic populations. To pulse label interphase cells with 3zP , petri dishes were rinsed with phosphatefree saline and cells were then grown for l-1.5 hr in “P-containing culture medium (see above). Cell Fractionation Mitotic or interphase cells were washed with cold PBS and then disrupted and fractionated by one of several possible procedures. Most commonly (for example, Figure 1). cell pellets (l-2 X 1 O6 cells) were suspended in 0.25 ml of cold buffer containing 0.01 M triethanolamine-HCI (pH 7.4), 0.01 M KCI. 0.0015 M MgC12. 0.0005 M phenylmethylsulfonyl fluoride (PMSF) and 0.005 M iodoacetamide. After swelling for 10 min on ice, cells were disrupted with 15-20 gentle strokes in a 0.5 ml glass-teflon Potter homogenizer (A. H. Thomas Inc.), and 0.25 ml of compensating buffer was added [O.Ol M triethanolamine-HOI (pH 7.4). 0.27 M KCI. 0.0015 M MgCl2. 0.0005 M PMSF and 0.005 M iodoacetamide]. Homogenates were then fractionated by centrifugation for 15 min at 40.000 rpm in a Beckman 40 rotor, yielding a pellet (~140) and a supernatant (~140). In some experiments, fractionation of mitotic cell homogenates was carried out directly in hypotonic homogenizing buffer. In other cases, a compensating buffer containing 1 M KCI was added to homogenates following hypotonic cell disruption. Generally, we ob-

served that increasing the ionic strength of the fractionation medium following the initial hypotonic homogenization resulted in a corresponding increase in the relative soluble proportion of lamins A and C (that is, the proportion in the ~140 fraction). The relative soluble fraction of the band containing lamin B was not increased by higher ionic strength, however, even when fractionation was performed in 0.5 M KCI. Direct homogenization of cells in isotonic buffer (without hypotonic swelling) gave the same fractionation pattern as our standard conditions (Figure 1). Detergent cell disruption was performed by resuspending a cell pellet in 0.5 ml of a buffer containing t % Triton X-100, 0.01 M triethanolamine-HCI (pH 7.4). 0.14 M KCI. 0.0015 M Mg&, 0.0005 M PMSF and 0.005 M iodoacetamide, and vortexing gently for 10 sec. Fractionation of detergent homogenates was performed as described above. We found it necessary to include protease inhibitors both during fractionation (PMSF and iodoacetamide) and during immunopreclpitation (trasylol, iodoacetamide and EDTA; see below) to avoid proteolytic degradation of the lamins. Lamin A is particularly sensitive to in vitro Proteolysis and is easily degraded to lower molecular weight species that migrate in the SDS gel region of lamins B and C. Previously, using antibodies raised against the P67 band of the rat liver Pore complex-lamina fraction, it was shown by Ouchterlony double-diffusion analysis that P67 is immunologically heterogeneous (Gerace et al., 1978). containing one component that strongly crossreacts with lamins A and C. Subsequent investigations (L. Gerace, A. Blum and G. Blobel. manuscript in preparation) have determined that this strongly crossreacting component of P67 is biochemically minor. The biochemically major component of P67 (which comprises >8090% of this eluted band) has a distinct Isoelectric point and cyanogen bromide peptide map from the other lamins. and is defined as lamin B (see Results). It is possible that the biochemically minor component of the rat liver P67 band (which strongly crossreacts with lamin A) is derived from lamin A by in vivo or in vitro proteolysis. lmmunopreclpitation Analysis We accomplished immunoprecipitation of the lamins with affinitypurified anti-lamin A antibodies using an indirect solid-phase procedure carried out in the presence of Triton X-100 and SDS. The preparation and characterization of anti-lamin A antiserum has been described by Gerace et al. (1978). Specific antibodies were affinitypurified from anti-lamin A antiserum (Gerace et al., 1978) using a column containing SDS-solubilized pore complex-lamina protein conjugated to Sepharose 48 (-1.5 mg protein per ml packed gel). Cells or cell fractions were first precipitated with 10% trichloroacetic acid (TCA) on ice. For samples obtained by detergent fractlonation, TCA precipitates were then extracted with 1 ml of cold 90% acetone. Next, samples were resuspended in a solution containing 0.5% SDS, 0.05 M triethanolamine-HCI (pH 7.4). 0.10 M NaCl and 0.002 M EDTA. sonicated briefly and boiled for 2 min. Typically, a volume of 0.5 ml of this solution was used to solubilize l-2 X 1 O6 cells, or fractions derived therefrom. Subsequently, the following solutions were successively added to 0.5 ml of SDS-containing sample solution, with vortex mixing between additions: 20% Triton X-l 00. 50 pL; 0.5 M iodoacetamide. 5 wL; Trasylol. 5 pL; and affinity-purified anti-lamin A antibodies (-0.5 Azao U/ml), 25 pL. After the addition of antibody, samples were first incubated for 2 hr at 37°C. They were then added to 25 pL (packed bead volume) of an immunoadsorbent consisting of rabbit anti-chicken IgG conjugated to Sepharose 48 (5 mg IgG per ml packed gel) and were incubated with agitation on an end-over-end mixer for 1 hr at 37°C. The immunoadsorbent beads were then washed four times in batch fashion with 1 ml of buffer containing 2% Triton X-l 00. 0.5% SDS, 0.05 M triethanolamine-HCI (pH 7.4), 0.10 M NaCl and 0.002 M EDTA. Following a final wash in 1 ml of 0.1 M triethanolamine-HCI (PH 7.4). immunoprecipitated protein was eluted from the immunoadsorbent by incubating the Sepharose beads for 5 min at 37°C with 60 flL Of a solution containing 6% SDS, 15% sucrose, 0.1 M triethanolammeHCl (pH 7.4) and 0.002 M EDTA. The solution recovered from the last step was given an addition of 5 PL of 1 M dithiothreitol and Was boiled for 3 min prior to analysis on an SDS gel.

Cell 286

SDS gel electrophoresis was accomplished on 7.5-15% linear gradient gels as described (Gerace et al., 1978). and fluorography (Banner and Laskey. 1974) or autoradiography was performed. To quantitate radioactivity contained in SDS gel bands, appropriate gel regions were excised from dried gels with a razor blade and were swelled in 150 pL of Hz0 (a slight excess over the amount imbibed by the gel) for 1 hr. Next, these samples were incubated with 1 ml of 90% NCS tissue solubilizer (Amersham) for 2-3 hr at 5O’C. Samples were then cooled and neutralized with 70 AL of glacial acetic acid. Radioactivity was determined in a scintillation counter after the addition of 9 ml of Aquasol(New England Nuclear). We calculate that we are at a reasonable level of antibody excess with our conditions of immunoprecipitation. In several experiments, the unbound protein sample remaining afler immunoprecipitation of 2 x lo6 interphase cells was then subjected to a second round of immunoprecipitation. Compared to the cpm in the lamin bands of the first immunoprecipitation step, we obtained an additional 5-20% of these cpm with the second immunoprecipitation. Two-Dimensional Gel Elsctrophoresis Samples were immunoprecipitated for two-dimensional gel analysis as described above, except that steps involving sample boiling were omitted and were replaced by 15 min incubations at 37°C. To elute immunoprecipitated protein from the immunoadsorbent beads for this procedure, we added 40 pL of a solution consisting of 9.5 M urea, 2% Nonidet P40 and 2% ampholines (0.4% 3-l 0 range, 1.6% 5-7 range) to 2.5 pL of packed gel beads, and in this mixture dissolved 25 mg of solid urea. This sample was then incubated for 30 min at room temperature, and the solution containing the eluted protein was removed from the Sepharose beads. First dimensional separation involved nonequilibrium pH gradient electrophoresis (O’Farrell et al., 1977) on slab gels having the dimensions 13 cm X 13 cm X 0.1 cm. The first dimensional gel was prepared with LKB ampholines as described by Ames and Nikado (1976). with modifications as described by Piperno. Huang and Luck (1977). Samples were applied to the anode (acidic) end of the gel and electrophoresed toward the cathode. Electrophoresis was for 4 hr at 2 watts constant power, with the general procedures described by Ames and Nikado (1976). At the end of this electrophoresis, sample lanes were cut from the first dimensional gel with a razor blade and were equilibrated with a solution consisting of 2% SDS, 0.1 M Tris-HCI (pH 6.8) and 0.01 M dithiothreitol by shaking for 15 min. These strips were then laid transversely on the top of a second dimensional 7.5-l 5% SDS gradient gel and subjected to electrophoresis under standard conditions. Acknowledgments

August

Banner. W. M. and Laskey. R. A. (1974). A film detection tritium-labeled proteins and nucleic acid in polyacrylamide J. Biochem. 46, 83-88.

23. 1979;

revised

October

26, 1979

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