Immunoaffinity Purification and Functional Characterization of ...

THE JOURNAL OF BIOLOGICAL. CHEMISTRY 8 1990 by The American Society for Biochemistry

Vol. 265, No. 21, Issue of July 25, pp. 12596-12601, 1990

Printed in U.S.A.

and Molecular Biology, Inc.

Immunoaffinity Purification and Functional Characterization Interphase and Meiotic Drosophila Nuclear Lamin Isoforms* (Received

Lin Lin From Stony

and Paul

for publication,

of February

9, 1990)

A. Fisher+

the Demrtment of Pharmacolo&al Brook: New York 11794-8651 -

Sciences.

Health

Three isoforms of a single nuclear lamin have been identified in Drosophila. Two, lamins Dm, and Dm2, are present during interphase and are apparently in equilibrium with each other in vivo. The third, lamin Dm,it, is found in cells that have undergone nuclear envelope breakdown, either during meiosis or mitosis. All three isoforms were purified under nondenaturing conditions using a novel technique of immunoaffinity chromatography and their in vitro activities were examined. Interphase lamins Dm, and Dmz can assemble into filaments at physiologic ionic strength; assembly is reversible upon addition of concentrated NaCI. Negative staining of filaments formed in vitro shows long, unbranched bundles approximately 20 nm in diameter. Addition of specific antilamin antibodies blocks in vitro assembly completely. In contrast with lamins Dml and Dmo, lamin Dm,,,it remains soluble at physiologic ionic strength. These observations are consistent with the notion that lamina disassembly in vivo is due, at least in part, to changes in properties of the lamins themselves.

Previous work from our laboratory has focused on the nuclear lamins. Only a single lamin gene has been identified (Gruenbaum et al., 1988) encoding only one primary translation product designated lamin Dm, (Smith et al., 1987). Lamin Dmo is processed rapidly in the cytoplasm to lamin Dml by what is apparently NHP-terminal proteolysis. This is followed by assembly into the nuclear envelope and further post-translational modification. Two isoforms, lamins Dml and Dmz, are found at significant steady-state levels in interphase nuclei. They arise through differential phosphorylation, can be distinguished by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE),’ and are in equilibrium with each other during normal growth (Smith et al., 1987). During both meiosis and mitosis, the Drosophila lamins redistribute throughout the cell and only a single lamin isoform, with an SDS gel mobility intermediate between those of lamins Dml and Drn? is found, this isoform is soluble in cell extracts and has been designated lamin Dmmit (Smith and Fisher, 1989). Lamins Dml, Dms, and Dm,,,it are all phosDrosophila

*This work was supported by Research Grants GM33132 and GM35943 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisemerit” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom correspondence should be addressed. Tel.: 516-4443067; Fax: 516-444-3218. ’ The abbreviation used is: SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

Sciences

Center,

State

University

of New

York,

phorylated to similar absolute levels (between 2 and 3 mol/ mol of protein); however, phosphoamino acid analyses revealed that while lamins Dml and Dma each contain both phosphoserine and phosphothreonine, lamin Dmmlt contains almost exclusively phosphoserine. Nuclear lamins are members of the intermediate filament protein family (for a review, see Nigg, 1989) and recently, Aebi et al. (1986) showed that the Xenopus nuclear lamina was composed of intermediate filament-like fibrils in situ. Moreover, Aebi et al. (1986) reported that purified interphase lamins A and C obtained from rat liver nuclei could reassemble into characteristic lo-nm filaments under appropriate conditions. Purification of rat liver lamins by Aebi et al. (1986) for in vitro assembly experiments entailed high salt/nonionic detergent extraction of lamins from 1 M KCl-washed nuclear envelopes followed by ion exchange chromatography in the presence of approximately 6 M urea. While effective for purification of interphase lamins from a highly enriched nuclear envelope fraction, this protocol was not suitable for purification of meiotic or mitotic lamin isoforms from much cruder total cell extracts. We thought it possible that immunoaffinity chromatography might provide the necessary stringency to purify both interphase and meiotic or mitotic nuclear lamin isoforms. In contrast with rat liver, Drosophila interphase lamins Dml and Dmp are extractable from nuclease-treated nuclei in solutions of high salt concentration alone (see e.g. Filson et al., 1985; McConnell et al., 1987); lamin Dmm,t can be obtained in large quantities from high-speed supernatant fractions of low ionic strength-early embryo extracts (Smith and Fisher, 1984; 1989).* In this article, we report the immunoaffinity purification of interphase lamins Dml and Dmz, and meiotic/ mitotic lamin Dm,,,, each in a single chromatographic step and without prolonged exposure to chaotropic agents or denaturants. Lamins Dml and DmP, purified in 0.5 M NaCl, apparently polymerize to form filaments when the salt concentration is reduced either by dilution or dialysis. In contrast, lamin Dmmit remains soluble at low ionic strength, thus substantiating the notion that lamina dissolution in uiuo results at least in part from specific changes in the properties of the lamins themselves. EXPERIMENTAL

PROCEDURES

Antibodies-Specific affinity purified antibody fractions were from Cappel Laboratories (Cochranville, PA). Affinity purified antilamin antibodies were prepared using a clone-encoded lamin+-galactosidase fusion protein (Gruenbaum et al., 1988) as recently described (Fisher and Smith, 1988). The specificity of this reagent has been documented * The soluble pool of lamin Dmmit present in low ionic strengthearly embryo extracts is derived from meiotic breakdown of the oocyte germinal vesicle during the final stage of Drosophila oogenesis (Smith and Fisher, 1989).

12596

Drosophila

Lamin

previously (see e.g. Smith et al., 1987; Gruenbaum et al., 1988; Smith and Fisher, 1989). Methods-Drosophila melanogaster (Oregon R, P2 strain) were maintained in mass culture according to Allis et al. (1977). SDSPAGE was according to Laemmli (1970) as modified (Fisher et al., 1982). Transfer of SDS-PAGE seoarated uroteins to nitrocellulose was passive; resulting immunoblom were probed with specific IgG fractions and bands of immunoreactivity were visualizedcolorimetricallv (Blake et al., 1984; Smith and Fisher, 1984). Calf alkaline phosphatase was glutaraldehyde conjugated to’ affinity purified goat anti-rabbit IgG (Avrameas, 1969); calorimetric detection was according to McGadey (1970). Cell Fractionation-Drosophila embryos were harvested, dechorionated, and stored frozen exactly as described previously (Fisher et al., 1982). To prepare extracts enriched in interphase lamins Dmi and Dmp, 6-18-h-old embryos were thawed and nuclei were prepared, digested with DNase I and RNase A. and extracted seauentiallv with 2% Triton X-100 and 0.5 N NaCl essentially as detailed previously (Fisher et al., 1982; McConnell et al., 1987). To facilitate efficient salt extraction of the Drosophila lamins, nuclease digestion was performed at 23 “C rather than at 37 “C (McConnell et al., 1987). The 0.5 M NaCl extract was used immediately as the source of lamins Dmi and Drn? for further purification. Extracts enriched for Drosophila lamin Dmm,, were prepared from 0-4-h-old embryos2 Frozen embryos were thawed and homogenized in the same buffers used for preparation of nuclei from older embryos. The homogenate was centrifuged first at 10,000 X g for 10 min and then at 100,000 X g in the SW27 rotor for 60 min. The supernatant from the 100,000 X g centrifugation (S-100) was used immediately as the source of lamin Dm,,,,, for-further purification. Preparation of Antilamin Protein A-Sepharose-Affinity purified anti-&osophila lamin IgG was adsorbed to Protein A-Sepharose CL4B (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) in 100 mM NaHP04, pH 6.8, by incubation for 2 h at room temperature. A ratio of about 1 mg of affinity purified IgG to 1 ml of hydrated Protein ASepharose was used. Glutaraldehyde (Fisher) was added to a final concentration of 0.01% and incubation was continued for 4-5 h at room temperature. Ethanolamine was then added to a final concentration of 200 mM and incubation at room temperature continued for 1 h more. After adsorption and conjugation, the antilamin-Protein A-Sepharose was poured into a column and the column was precycled bv washing first with 20 mM Tris-HCl. oH 8.1. 500 mM NaCl. 0.1% Briton X-TOO, 5 mM EDTA (Equilibration Buffer), and then with 50 mM glycine/HCl, pH 2.3, 500 mM NaCl, 0.1% Triton X-100 (Elution Buffer) plus 0.02% SDS alternately, at least twice. Once prepared and standardized, antilamin Protein A-Sepharose columns were stable and could he reused repeatedly without evidence of deterioration. Immunoaffinity Purification of Drosophila Nuclear Lamin Isoforms-Before being subjected to immunoaffinity chromatography, Drosophila extracts prepared as described above were made 1 mM in phenylmethylsulfonyl fluoride. Lamins from these extracts were batch-adsorbed to antilamin Protein A-Sepharose by incubation overnight at 4 “C. The column was then washed extensively with Equilibration Buffer and purified lamins were recovered from the antilamin affinity column with Elution Buffer. After elution, the solution containing the lamins was immediately neutralized with Na,HP04 added to a final concentration of 50 mM, aliquoted, frozen in liquid nitrogen, and stored at -70 “C. Additional details are provided in the figure legends. Electron Microscopy-For negative staining, samples were adsorbed onto copper grids which had been coated with a thin carbon film (50-75 A) supported by a perforated Formvar film. Just before loading, grids were treated with a 200 rg/ml solution of cytochrome c. After sample adsorption for 30 s, grids were rinsed with doubledistilled water and negatively stained with 1% uranyl acetate. Electron microscopy was on a Jeol 1200 EX electron microscope at 80 kV. RESULTS

Immunoaffinity Purification of Lamins Dm,, Dma, and Dm,it-Lamin Dm,,,it is abundant and soluble in low ionic strength extracts of Drosophila early embryos.’ An S-100 fraction prepared from 0-4-h-old Drosophila embryos (“Experimental Procedures”) served as the source of lamin Dmmit for immunoaffinity purification. To immunoaffnity purify interphase Drosophila lamins Dm, and Dm,, it was first

12597

Polymerization

necessary to solubilize them from nuclei under nondenaturing conditions. To accomplish this, nuclei were purified from 618-h-old Drosophila embryos, digested with nucleases at 23 “C, and extracted with 2% Triton X-100 as described (“Experimental Procedures”). The residual nuclear pellets were then extracted with a solution of 0.5 M NaCl which resulted in the solubilization of about 75% of the lamin present (data not shown). Analysis of interphase lamins Dml and Dms immunoaffinity purified from the 0.5 M NaCl extract of Drosophila nuclei is shown in Fig. 1, A and B, lanes a, and in Fig. 1C. Analysis of immunoaffinity purified lamin Dm,,, is shown in Fig. 1, A and B, lanes 6. Fig. 1, A and C, show Coomassie Blue-stained gels; Fig. 1B shows an immunoblot of a gel run in parallel to the one shown in Fig. L4 that was probed with affinity purified antilamin antibodies. Interphase lamins Dml and Dmn appeared nearly homogeneous after a single step of immunoaffinity

chromatography;

this

is particularly

evident

from

the

minigel analysis shown in Fig. 1C. In contrast, immunoaffinity purified lamin Dmmit fractions were reproducibly contaminated by a major polypeptide with an apparent mass of 90 kDa. This species is designated by the downpointing arrowhead in Fig. lA, lane b. Examination of the parallel immunoblot (Fig. lB, lane b) showed that this 90-kDa polypeptide was apparently not reactive with the antilamin antibodies used for immunoaffinity purification. Also apparent in Fig. lA, lane b, are a number of minor polypeptides, all smaller than lamin Dm,i,. Within the sensitivity limits of the parallel immunoblot analysis (Fig. lB, lane b), it appeared that those polypeptides were all reactive with the antilamin antibodies used for immunoaffinity purification, consistent with the notion that these minor species were proteolytic breakdown products of lamin Dmmi,. Assembly of Drosophila Nuclear Lumins into Filaments in Vitro-To investigate the potential for immunoaffinity purified Drosophila lamins to form filaments in vitro, samples in buffer containing 0.5 M NaCl were dialyzed against buffer containing 0.1 M NaCl. Samples were maintained at 23 “C and at various times after the start of dialysis, aliquots were taken, separated into supernatant and pellet fractions by microcentrifugation, and the fractions analyzed by SDSPAGE and Coomassie Blue staining. The results of one such experiment, performed with immunoaffinity purified interphase lamins Dml and Dm2 are shown in Fig. 2A. In a control experiment, lamins Dml and Dmz were dialyzed against buffer containing 0.5 M NaCl such that the final NaCl concentration remained constant throughout (Fig. 2B). Upon dialysis against 0.1 M NaCl, there was clearly a change in the behavior of lamins Dml and Dm,. After as little as 30 min, a majority of the lamin was detectable in the pellet fraction (Fig. 2A, lanes c and d) whereas before dialysis, lamins were found only in the supernatant (Fig. 2A, lanes a and b). After 2 h, most of the lamin was recovered in the pellet fraction (Fig. 2A, lanes g and h). In contrast, dialysis of lamins Dml and Dmz against buffer containing 0.5 M NaCl had no effect on the solubility of the lamins over the duration of this experiment (Fig. 2B). To characterize further the apparent in vitro assembly of immunoaffinity purified lamins Dml and Dmp, the products of the assembly reaction were examined by electron microscopy after negative staining. Before dialysis, no higher order structures were seen (not shown). This was consistent with the observation that lamins Dml and Dmz remained in the supernatant fraction following microcentrifugation under these conditions (see Fig. 2). When the sample was dialyzed against 0.1 M NaCl for 30 min before being processed for

12598

Drosophila

A

B a

b

Lamin Polymerization

C a

A

b

a a

b

bc

d

cd

B

v 76~ 74-

--

a

m-

bc

def

+75

FIG. 1. Immunoaffinity purified Drosophila lamins Dml, Dmz, and Dm,,,; SDS-PAGE and immunoblot analysis. A, Coomassie Blue-stained gel; B, immunoblot of a gel run in parallel to the one shown in A and probed with affinity purified antilamin antibodies at a final concentration of about 0.2 @g/ml. SDS-7% PAGE was on 20.cm long, 1.5.mm thick gels to better resolve the different Drosophh lamin isoforms. Lanes a were loaded with lamins Dml and DmL immunoaffinity purified together from a 0.5 M NaCl extract of nuclease-treated nuclei (nuclear salt extract; “Experimental Procedures”); lanes b were loaded with lamin Dm,,, immunoaffinity purified from an early embryo S-100 (“Experimental Procedures”). Gels were loaded as follows, quantities refer to amounts of total protein loaded in a given lane; panel A, lane a, 500 ng; panel A, lane b, 1500 ng; panel R, lane a, 200 ng; panel B, lane b, 100 ng. One-dimensional chymotryptic peptide map analysis (Smith et al., 1987) confirmed that the major immunoreactive proteins specifically recovered from antilamin Protein A-Sepharose columns were indeed lamins (not shown). C, Coomassie Blue-stained miniael. SDS-8% PAGE of fractions from immunoaffinity purification of lamins Dm, and Drn? was on a 4-cm long, 0.75-mm thick minigel. Samples loaded in each lane were as follows; lane a, 5 units (Fisher et al., 1982) of nuclear salt extract; lane b, an equivalent amount of the nonadsorbed fraction after incubation of the nuclear salt extract with antilamin Protein ASepharose; lane c, an equivalent amount of the final immunoaffinity column wash prior to elution of lamins Dml and Dm2; lane d, lamins Dml and DmL (not resolved) eluted from the antilamin Protein ASepharose column, overloaded to demonstrate purity. Parallel immunoblot and one-dimensional chymotryptic peptide map analyses confirmed identification of the major Coomassie Blue-stained polypeptide in panel C’, lane d, as lamin (not shown). Arrows to the left of panel A designate the migration positions of lamins Dm, (74) and Drn,! (76); arrow to the right of panel B designates the migration position of lamin Dm,,, (7.5). Unlabeled arrow to the right of panel C designates the migration position of Drosophila lamins on this 8% polyacrylamide minigel. Unlabeled bidirectional arrows between panels A and B designate the migration positions of marker proteins; from top to bottom, they are myosin heavy chain (200 kDa), pgalactosidase (116 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), and ovalbumin (46 kDa).

electron microscopy, the results shown in Fig. 3 were obtained. Consistent with a shift from the supernatant to the pellet fraction after microcentrifugation (Fig. 2A), extended polymeric structures were observed by electron microscopy (Fig. 3). Occasionally, uniform unbranched fibers were seen (Fig. 3, A and B). More often, structures were relatively heterogeneous in appearance and were clearly bundles or aggregates of thinner fibrils (Fig. 3, C and D). Similar aggregates were

FIG. 2. Effect of changing NaCl concentration on the solubility of immunoaffinity purified lamins Dml and Dmz. Coomassie Blue-stained minigels; A, a solution of immunoaffinity purified lamins Dm, and Dm*, total protein concentration of 40 Kg/ml, in 0.5 M NaCl plus 50 mM glycine, 50 mM Na,HPO,, pH 7 (final), was subjected to open dialysis at 23 “C using Millipore 0.05-b glass filter membranes. Dialysis was against 0.1 M NaCl plus 20 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, and at various time points, aliquots were withdrawn and separated into supernatant and pellet fractions by microcentrifugation in a Fisher Microcentrifuge for 30 min at 4 “C. After microcentrifugation, supernatants and pellets were separately SDS-denatured and subjected to SDS-PAGE on 7% polyacrylamide minigels. Gels were stained with Coomassie Blue, destained, dried, and photographed. Lanes a, c, e, and g, sur ornatant fractions; lanes b, d, f, and h, corresponding pellet fractions. Lanes a and b, before dialysis; lanes c and d, aliquot taken 30 min after the onset of dialysis; lanese and f, aliquot taken 60 min after the onset of dialysis; lanes g and h, aliquot taken 120 min after the onset of dialysis. B, experiment was identical to that shown in A except that dialysis was against 0.5 M NaCl plus 20 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride. Lane designations are as in A. Unlabeled arrows to the right of panels A and B designate the migration positions of Drosophila lamins on these polyacrylamide minigels.

reported by Aebi et al. (1986) although in their experiments, a fine paracrystalline pattern could be discerned within the aggregates. No such pattern was discernible in our experiments. It was suggested to us by Dr. Joseph Wall (Brookhaven National Laboratory) that initiation of lamin polymerization reactions by dilution rather than dialysis might lead to formation of more uniform structures. That this in fact proved to be the case is demonstrated by the electron micrographs in Fig. 4. When samples were examined 50 min after dilution of interphase lamins Dml and Dmp from 0.5 to 0.1 M NaCl, polymeric structures were seen. However, unlike the majority of structures seen after dialysis (Fig. 3), these were all of relatively uniform caliber (approximately 20 nm) and were unbranched (Fig. 4, A and B). Examination at higher magnification suggested that the structures formed were bundles of 2-4 thinner filaments (Fig. 4C). As with dialysis, formation of filaments after dilution as judged by transmission electron microscopy, correlated with sedimentation of lamins by microcentrifugation (not shown; but see Figs. 5 and 6). A number of additional experiments were performed by dilution using microcentrifugation to monitor assembly. These results are shown in Fig. 5. Assembly was dependent on the final salt concentration after dilution (Fig. 5A), was protein concentration-dependent (Fig. 5B), and

Drosophila

Lamin Polymerzation

was reversible if the salt concentration was returned to 0.5 M by the addition of concentrated NaCl (Fig. 5C). Specific Antilamin Antibodies Block in Vitro Assembly of Filaments from Immunoaffinity Purified Lamins Dm, and Dmp-The effect of affinity purified antilamin antibodies on the polymerization of lamins Dm, and Dms is shown in Fig. 6. Addition of approximately equimolar amounts of antilamin IgG completely blocked in uitro lamin polymerization, both as judged by microcentrifugation (Fig. 6, compare lanes a and b with c and d) and transmission electron microscopy (not shown). In contrast, nonimmune IgG did not interfere with in vitro lamin polymerization as judged by microcentrifugation, even when added in considerable molar excess relative to the lamins (Fig. 6, compare lanes e-h with lanes a and b). Incubation of Immunoaffinity Purified Lamin Dm,;, at Low

FIG. 3. Transmission electron microscopic analysis of lamins polymerized in vitro after dialysis to reduce the NaCl concentration. A 40.@g/ml solution of immunoaffinity purified lamins Dm, and DmL in buffer containing 0.5 M NaCl was dialyzed against buffer containing 0.1 M NaCl as described in the legend to Fig. 2. After 30 min of dialysis, samples were withdrawn and processed for electron microscopy as described (“Experimental Procedures”). The bar in panel A designates 1 Km; the bars in panels B and C each designate 200 nm; the bar in panel D designates 100 nm.

WC. 4. Transmission electron microscopic analysis of lamins polymerized in vitro after dilution to reduce the NaCl concentration. A 200&ml solution of lamins Dml and Dm, in buffer containing 0.5 M NaCl was diluted 5-fold in buffer alone such that the final NaCl concentration was 0.1 M. After incubation for 50 min on ice, samples were withdrawn and processed for electron microscopy as described (“Experimental Procedures”) and exactly as for the samples shown in Fig. 3. The bar in panel A designates 500 nm; the bar in panel B designates 200 nm; the bar in panel C designates 50 nm.

12599

Ionic Strength Does Not Lead to Precipitation or AssemblyIn contrast with results obtained with immunoaffinity purified interphase lamins Dm, and Dm2, immunoaffinity purified lamin Dmmit remained soluble at 0.1 M NaCl regardless of whether dilution or dialysis was used to achieve that final NaCl concentration. The results of one such experiment, performed by dialysis so as to maintain the concentration of lamin Dmmi, as high as possible, are shown in Fig. 7. Over the time course of this experiment, there was no change in the distribution of lamin Dmmi, after microcentrifugation. Examination by transmission electron microscopy, of a sample taken at the final time point of dialysis, failed to detect any higher order structures (not shown). DISCUSSION

Immunoaffinity chromatography has proven effective for purification of both meiotic (mitotic) and interphase Drosophila nuclear lamin isoforms under nondenaturing conditions. As far as we are aware, this is the first reported purification of a meiotic or mitotic nuclear lamin isoform from any species. Moreover, although interphase nuclear lamins have previously been purified from rat liver by Aebi et al. (1986), the protocol used involved ion exchange chromatography in the presence of 6 M urea. By comparison, our procedure is both rapid and does not require prolonged exposure of lamins to denaturants such as urea. Immunoaffinity chromatography using specific antibodies covalently conjugated to Protein A-Sepharose was originally introduced by Gersten and Marchalonis (1978). In their protocol, dimethylsuberimidate was used as the cross-linking reagent. Although the applicability of other reagents has since been reported (see e.g. Schneider et al., 1982), the use of glutaraldehyde for preparing specific IgG-Protein A-Sepharose immunoaffinity chromatography matrices is, to the best of our knowledge, novel. Indeed, in their report, Gersten and Marchalonis (1978) indicated that for unspecified reasons, glutaraldehyde was unsuitable. Our success with glutaraldehyde was therefore, unanticipated. Nevertheless, in numerous control experiments, glutaraldehyde, used at very low concentrations (O.Ol-0.05%), was found to be effective both in terms of coupling efficiency and preservation of antibody reactivity

Drosophila

12600 A

s

P a

abed

L-

B

b

c

a

b

c

d

e

f

9

h

S

P

s

p

s

P

s

P

d

*

CL--

P

S

a


b

c

a

bc

a

b

c

d

C

FIG. 6. Effect of antilamin and irrelevant antibodies on the in vitro polymerization of lamins. Immunoblots probed with affinity purified antilamin antibodies as in Fig. 1B. SDS-7% PAGE was on minigels essentially as in Fig. 1C. A 200-pg/ml solution of lamins Dm, and Dmp in buffer containing 0.5 M NaCl was diluted with 20 mM Tris-HCl, pH 7.5, to a final NaCl concentration of 0.1 M. After dilution, samples were incubated for 60 min on ice and then subiected to microcentrifucration followed bv SDS-PAGE. transfer of proteins to nitrocellulose, z&d immunoblot analysis. Supernatant and pellet fractions are designated s and p, respectively. Immunoblot analysis was used for this experiment to increase sensitivity relative to that which could be obtained with Coomassie Blue staining. Before dilution, additions to the lamin solutions were as follows; lanes a and b, none; lanes c and d, approximately equimolar amounts, relative to the lamins, of affinity purified rabbit antilamin IgG; lanes e and f, approximately equimolar amounts of irrelevant rabbit IgG; lanes g and h, approximately a 5-fold molar excess of irrelevant rabbit IgG. The downpointing arrowheads in lanes c, e, and g indicate the IgG heavy chain band recognized in the immunoblot analysis due to primary reactivity with the calf alkaline phosphatase-conjugated goat anti-rabbit IgG used as the secondary reagent for immunodetection (“Experimental Procedures”). Unlabeled arrow to the right of the blot designates the migration position of Drosophila lamins on this polyacrylamide minigel.

a b FIG. 5. In vitro polymerization of lamins following dilution is dependent on the final NaCl concentration, the final protein concentration, and is reversible upon return of the sample to high NaCl concentration. Coomassie Blue-stained minigels; SDS7% PAGE. A, aliquots of a 200.pg/ml solution of lamins Dm, and Dm,, were diluted 5-fold with solutions of varying NaCl concentrations, incubated on ice, and then subjected to microcentrifugation followed by SDS-PAGE analysis as described in the legend to Fig. 2. Supernatant and pellet fractions are designated s and p, respectively. The final NaCl concentrations were as follows; lanes a, 0.075 M; lanes b, 0.15 M; lanes c, 0.3 M; lanes d, 0.5 M. B, a 200-pg/ml solution of lamins Dml and Drn? in buffer containing 0.5 M NaCl was diluted in buffer such that the final NaCl concentration was 0.1 M and the final protein concentration was varied. Incubation after dilution, microcentrifugation, and SDS-PAGE were as in A. Supernatant and pellet fractions are designated s and p, respectively. The final protein concentrations were as follows; lanes a, 40 pg/ml; lanes b, 16 pg/ml; lanes c, 8 pg/ml. C, a 200-pg/ml solution of lamins Dml and Dmt in buffer containing 0.5 M NaCl was diluted 5-fold with buffer to a final NaCl concentration of 0.1 M and incubated on ice for 60 min. One aliquot was taken for microcentrifugation and SDS-PAGE analysis at this point; lane a, supernatant; lane b, pellet. The NaCl concentration of a second aliquot was returned to 0.5 M by addition of a small volume of a concentrated NaCl stock solution. After an additional 120 min, this second aliquot was taken for microcentrifugation and SDS-PAGE; lane c, supernatant; lane d, pellet. Unlabeled arrows to the right of each panel designate the migration positions of Drosophila lamins on these polyacrylamide minigels.

after coupling.” We therefore recommend that its use be considered, particularly when other cross-linking reagents prove unsuitable. Interphase Drosophila nuclear lamins Dm, and Dmp were purified to near-homogeneity in a single step of immunoaffinity chromatography. In contrast, while highly purified, the ” L. Lin

and P. A. Fisher,

unpublished

data.

c

d

e

f

g

h

ij

FIG. 7. Immunoaffinity purified lamin Dmmit remains soluble after dialysis against buffer of low NaCl concentration. Coomassie Blue-stained minigel. A 40-pg/ml solution of lamin Dmmit was dialyzed buffer containing 0.1 M NaCl and aliquots taken _ against at various times after the beginning of dialysis subjected to microcentrifueation and SDS-PAGE exactlv as described in the legend to Fie. 2.4. Lanes a, c, e, g, and i, supernaiant fractions; lanes b,-d, j, h, a& j, corresponding pellet fractions. Lanes a and b, aliquot taken before the onset of dialysis; lanes c and d, aliquot taken 15 min after the onset of dialysis; lanes e and f, aliquot taken 30 min after the onset of dialysis; lanes g and h, aliquot taken 60 min after the onset of dialysis; lanes i and;, aliquot taken 120 min after the onset of dialysis. Downpointing arrowheads in lanes a, c, e, g, and i designate p90 as in Fig. lA, lane b. Unlabeled arrow to the right of the gel designates the migration position of Drosophila lamins on this polyacrylamide minigel.

lamin Dmmit fraction prepared similarly was clearly not homogeneous. Rather, it was reproducibly contaminated by an approximately 90-kDa polypeptide, hereafter designated p90. p90 was present in substoichiometric amounts relative to lamin Dmmit and remained in the lamin Dm,,,it fraction, even after repeated rounds of immunoaffinity chromatography. Based on immunoblot analysis, p90 was unreactive with the

Drosophila


antilamin antibodies used to prepare the immunoaffinity chromatography matrix. There are undoubtedly a number of plausible explanations for the appearance of p90 in immunoaffinity purified lamin Dm,,,it fractions. However, one which we think makes further investigation of p90 particularly worthwhile is the possibility that p90 is present in the early embryo in some sort of biologically meaningful complex with lamin Dm,it. This explanation is perhaps made more likely by the observation that when early embryo extracts are denatured in SDS before immunoaffinity chromatography (performed in the presence of a &fold excess of Triton X-100 relative to the SDS; see Smith et al., 1987), lamin Dmmit is obtained free of p90.3 Aebi et al. (1986) reported that purified rat liver lamins A and C were able to form filaments under appropriate conditions in vitro. The in vitro assembly Drosophila lamins Dml and Dmz is therefore not entirely novel. However, the Drosophila lamins are thought to be members of the lamin B subfamily

(see Nigg, 1989; Smith

et al., 1987; Gruenbaum

et

al., 1988). They are therefore, the first B-type lamins for which in uitro assembly has been demonstrated. In this context, we think it noteworthy that Aebi et al. (1986) observed single lo-nm filaments after in vitro assembly of rat liver lamins

A and C, whereas

we observed

what

are apparently

bundles of about 20 nm in diameter. It remains to be determined whether these differences reflect meaningful differences between rat liver and Drosophila, between A-type lamins and B-type lamins, or rather, are indicative of technical differences

in lamin

purification

and/or

in vitro

assembly

conditions. The fact that specific antilamin antibodies can be used to block polymerization of immunoaffinity purified lamins Dmi and Dmz suggests that site-specitic antibody probes may be useful in deducing the intermolecular interactions involved in lamina assembly in uitro. This could be accomplished either by identification and mapping of monoclonal antilamin antibodies with the ability to block assembly, or generation and identification of polyclonal antipeptide antibodies with similar properties. That such probes could ultimately be employed to study nuclear envelope assembly in uiuo is suggested by the results of Benavente and Krohne (1986). They showed that microinjection of polyclonal antilamin antibodies into mitotic PtKz cells blocked normal nuclear reformation at the end of mitosis. The ability of immunoaffinity purified lamins Dml and

DmP to form filaments

and the failure

of lamin

Dm,,,it to do

so under identical conditions provides the first direct evidence that lamina disassembly in uiuo is due at least in part to specific changes in the lamins themselves. Both in vertebrates (see e.g. Ottaviano and Gerace, 1985) and in Drosophila (Smith and Fisher, 1989) lamina disassembly and reassembly in uiuo have been correlated with changes in patterns of lamin

phosphorylation. It might therefore be of interest to determine whether nonspecific phosphatases and/or kinases can alter the ability of immunoaffinity purified Drosophila lamins to form filaments. Ultimately, the in uitro properties of purified Drosophila lamins might facilitate design of assays to identify and purify specific phosphatases and/or kinases involved in regulating

lamina

dynamics

in uiuo.

Acknowledgments-It is a pleasure to acknowledge David Colflesh for performing electron microscopic analyses, Maeve McConnell for expert technical assistance, and Toni Daraio for help in preparing the manuscript.

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