Purification and Immunological Detection of Pea Nuclear Intermediate Filaments: Evidence for Plant Nuclear Lamins

Journal of Cell Science 103, 407-414 (1992) 407 Printed in Great Britain © The Company of Biologists Limited 1992

Puriﬁcation and immunological detection of pea nuclear intermediate ﬁlaments: evidence for plant nuclear lamins

A. K. McNULTY and M. J. SAUNDERS*

Biology Department, University of South Florida, Tampa, Florida 33620, USA *To whom correspondence should be addressed

Summary

A major structural component of the inner face of the conclude that plant cells contain this ancestral class of nuclear envelope in vertebrates and invertebrates is the intermediate filaments in their nuclei and that regula- nuclear lamina, an array of 1-3 extrinsic membrane pro- tion of nuclear envelope assembly/disassembly and mito- teins, lamins A, B and C. These proteins are highly sis in plants may be similar to that in animal cells. homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization Key words: nuclear lamins, intermediate filaments, Pisum sativum of putative plant lamin proteins from pea nuclei. We (pea).

Introduction attached to the membrane (Gerace and Blobel, 1980; Otta- viano and Gerace, 1986; Ward and Kirshner, 1990). The The nuclear envelope of eukaryotic cells is responsible for nuclear envelope fragments into small vesicles as the chro- segregating the nucleoplasm from the cytoplasm, regulat- mosomes condense and the mitotic spindle forms. During ing transport through nuclear pores, maintaining chromatin telophase, the lamins become dephosphorylated, associate organization in interphase, and disassembling and reassem- with chromatin, polymerize into a fibrous network and the bling during mitosis. A major structural component of the nuclear envelope re-forms around the decondensed chro- inner face of the nuclear envelope in vertebrates and inver- matin (Gerace et al., 1984; Glass and Gerace, 1990). Thus tebrates is the nuclear lamina, an array of one to three prin- the nuclear lamins are hypothesized to be responsible both ciple extrinsic membrane proteins, lamins A, B and C (Mr for the structural integrity and organization of the nucleus 70,000-60,000), which together form a fibrous network (see during interphase and function as a key regulator of the cell Abei et al., 1986; Gerace, 1986). cycle. Lamins are highly homologous in sequence and structure One to five lamins have been described from a wide vari- to intermediate filament (IF) proteins (Fisher et al., 1986) ety of vertebrates and invertebrates including humans, rats, and have recently been classified as type V IF. Biochemi- mice, birds, frogs, clams and fruit flies (see Krohne and cal and molecular analyses have indicated that lamins A Benavente, 1986; Osman et al., 1990). There has been some and C are more closely related to each other than to lamin speculation and initial evidence about the presence of inter- B (Krohne and Benavente, 1986; McKeon et al., 1986; mediate filaments, lamin-type proteins and cell cycle-regu- Osborn and Weber, 1986). Structurally, all IF proteins share lated phosphorylation events in plants. However, to the best a common central a -helical rod domain with long heptad of our knowledge, nuclear lamin proteins have never been repeat patterns of hydrophobic residues. At the amino and isolated and described as such in plants. One would imag- carboxyl terminal ends of the rod domains, all IF classes ine that a similar nuclear structure and regulation would share two highly conserved sequences. The rod portion is exist in plant cells to those in other eukaryotic organisms. flanked by variable, non-a -helical head and tail domains It has been proposed that nuclear lamins are the earliest (Geisler and Weber, 1982; Steinert et al., 1985; McKeon et type of intermediate filaments to have evolved (Osman et al., 1986; Fisher et al., 1986). al., 1990) and therefore it may be, that of all the types of During interphase, lamin B binds to a receptor on the intermediate filaments, the nuclear lamins will be found in inner nuclear envelope and lamins A and C bind to both plants. lamin B and decondensed chromatin. During prophase of Antigenically-determined intermediate filaments that are mitosis, it is hypothesized that a complex cascade of phos- related to type III IF (Dawson et al., 1985; Goodbody et phorylation events culminates in phosphorylation of the al., 1989; Hargreaves et al., 1989) and type II IF (Ross et lamins, releasing lamins A and C, while lamin B remains al., 1991) have been reported in the cytoplasm of higher 408 A. K. McNulty and M. J. Saunders plant cells. Monoclonal antibodies to plant nuclear matrix buffer B. The pellet was assayed for purity by microscopic analy- proteins recognize several nuclear proteins that are sis. immunologically related to intermediate filaments (Beven To isolate the nuclear lamina, the purified nuclei were resus- et al., 1991). Monoclonal antibodies to a high-salt Triton pended in lamina isolation buffer (lysis buffer B minus 2-mer- X-100 insoluble fraction (which is characteristic of IF) from captoethanol and spermine) and passed through a 25-gauge needle Chlamydomonas produces both a diffuse cytoplasmic fluo- 3 times. Nuclei were then incubated for 1 h with 250 mg/ml DNAase I and 250 mg/ml RNAase I. Samples were then mixed at rescence as well as a perinuclear fluorescence in onion root 0oC with an equal volume of high salt buffer (HSB; 20 mM Tris- tips (Parke et al., 1987). HCl, pH 8, 4 M NaCl, 2 mM EDTA and 2 mM DTT) and lay- Mitosis promoting factor (MPF), with its associated ered over a cushion of 15% sucrose in HSB. The nuclear lamina p34cdc2 kinase activity, has been shown to phosphorylate was pelleted at 20,000 g for 30 min in a Beckman J-21 centrifuge. lamins (Dessev et al., 1991) and purified p34cdc2 induces The lamina pellet was washed once with 5 mM Tris-HCl, pH 7.2. lamina disassembly in isolated nuclei (Peter et al., 1990). This protein has been immunologically detected in both Solubilization of plant nuclear lamins higher and lower plants including Arabidopsis, oats, Lamins were purified from pea nuclei as described above. One Chlamydomonas and brown and red algae (John et al., half of the final lamin pellet was treated with 0.02 M NaOH and 1989). In addition, p34 protein kinase and a cdc2 gene centrifuged at 140,000 g for 15 min. The initial and final pellets homologue have been described in pea (Feiler and Jacobs, and supernatants were run on a 7.5-15% SDS PAGE gel as 1990). described below. In this paper we present biochemical and immunological evidence for the presence and localization of 4 lamin pro- Protein electrophoresis teins in Pisum sativum (pea) nuclei. These are antigenically Protein was assayed by a modified Lowry procedure (Markwell similar to both animal lamins and intermediate filaments, et al., 1978). Lamina pellets were resuspended in 1´ Laemmli sample buffer (Laemmli, 1970) and boiled for 3 min. The super- and can be solubilized from the nuclear envelope by NaOH natants were loaded onto either 10% or 7.5 to 15% acrylamide treatment. However they appear to be distributed throught gradient SDS-PAGE gels on an equal protein basis. Gels were the nuclear matrix and not restricted to the nuclear enve- electrophoresed at 40 mA constant current. lope. Immunoblotting and immunostaining Materials and methods For immunoblotting, the protein was electrophoretically transferred to IMMOBILON PVDF membranes (Millipore) from acry- Plant material and growth conditions lamide gels at 100 V for 1 h. Blots were then either stained with Pea (Pisum sativum cv. milk and honey) seeds were sown in ver- 0.1% Coomassie Brilliant Blue or immunostained. miculite and grown in the dark at 22oC for 2 weeks. At this point For immunostaining, blots were blocked overnight at 4oC with etiolated leaves were harvested for lamina isolation. In addition, 10% horse serum in Tris-buffered saline + Tween 20 (TBST pea seeds were germinated on damp paper towels for 4 days and buffer; 10 mM Tris-HCl, pH 9.5, 150 mM NaCl, 0.05% Tween the root tip excised for immunocytochemistry. 20). After blocking, the blots were incubated with either anti-intermediate filament (anti-IF) antibody in TBST + 1% horse serum Rat and Hela cell lamina isolation or with anti-human lamin B signal sequence antibody in Tris- Rat liver lamins were isolated according to Dwyer and Blobel buffered saline (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.02% (1976). In addition, rat and HeLa cell lamin/nuclear pore extracts sodium azide) + 1% horse serum for 1.5 h at room temperature. were provided by Dr. Chaudhary, The Rockefeller University. Anti-IF was diluted 1:50 and anti-human lamin B was diluted 1:800. Secondary antibodies (diluted 1:4000) were either goat anti- Pea protoplast, nuclei and lamina isolation mouse IgG conjugated to alkaline phosphatase (for anti-IF) or goat Protoplasts were isolated from 3-4 g of etiolated pea leaves by anti-rabbit IgG conjugated to alkaline phosphatase (for anti-human incubation for 8 h at room temperature in protoplast isolation lamin B), and were applied for 1 h. Color was developed by incu- buffer (0.6 M sucrose, 0.07 g/100 ml cellulase, 0.06 g/100 ml bation of the blot in 3.3 mg nitroblue tetrazolium and 1.7 mg 5- drieslase, 0.05 g/100 ml macerace, 0.07 g/100 ml spermine, 10 bromo-4-chloro-3-indolyl phosphate in 10 ml of alkaline phos- mM 2-[N-morpholino]ethanesulfonic acid, pH 5.3, 0.04% v/v 2- phatase buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM mercaptoethanol, 10 mM NaCl and 10 mM KCl. This solution MgCl2). was then filtered through a 100 µm wire mesh (Newark Wire Cloth Controls were run with bovine serum albumin (BSA), rat liver Company). and HeLa cell lamin/pore complex extracts (prepared as described Protoplasts were pelleted at 1000 rev./min for 10 min in an IEC above and gift of Dr. Chaudhary, Rockefeller University). HN-SII centrifuge fitted with an IEC model 215 swinging bucket rotor (International Equipment Company). The pellet, containing Antibody preparation the protoplasts, was resuspended in 3 ml of lysis buffer (0.25 M Mouse-mouse hybridoma cells that secrete a monoclonal IgG1 sucrose, 5 mM Tris-HCl, pH 7.2, 0.75 mM MgCl2, 0.5% Triton antibody that reacts with all classes of intermediate filaments X, 0.1 mM spermine, 0.04% v/v 2-mercaptoethanol, 2.5 mM (American Type Culture Collection TIB 131) were cultured (Pruss EDTA and 2.5 mM DTT) and sedimented for 10 min at 1000 et al., 1981). The supernatant was used without further purifica- rev./min. Protoplast lysates were layered over an equal volume of tion. Anti-human lamin B (made against a synthetic peptide lysis buffer (0.4 M sucrose, 5 mM Tris-HCl, pH 7.2, 0.35 mM derived from the human lamin B signal sequence) polyclonal IgG MgCl2, 0.04% v/v 2-mercaptoethanol, 2.5 mM EDTA, 0.1 mM antibody (rabbit) was a gift from Drs Chaudhary and Blobel, The spermine and 2.5 mM DTT) and then pelleted at 1800 rev./min Rockefeller University. Control antibodies used were a mouse for 10 min. The pellet (containing nuclei) was then resuspended monoclonal anti-yeast tubulin (Sigma) and a rabbit polyclonal in lysis buffer (minus Triton X) and resedimented through lysis anti-prunin (gift of M.E. Colter, University of South Florida.) Plant nuclear lamins 409

Immunocytochemical localization of anti-IF and anti-lamin B in pea nuclei Plant lamins were localized by light microscopy using indirect immunofluorescence or electron microscopy (EM) using an indirect immunogold labeling procedure. Briefly, protoplasts, purified pea nuclei or the purified lamina fraction were fixed and perme- abilized in 1% glutaraldehyde, 0.5% Triton X-100 in 0.2 M phosphate buffer (pH 7.2). For light microscopy, protoplasts were attached to coverslips using poly-L-lysine, air dried and extracted in cold methanol for 5 min. All preparations were then sequen- tially incubated in 5% horse serum in 0.1% BSA-Tris for 20 min, anti-IF or anti-lamin B in 0.1% BSA-Tris for 2 h and washed 3´ 10 min in 0.1% BSA-Tris. Control nuclei were processed with either no primary antibody, a mouse monoclonal anti-tubulin or a rabbit polyclonal anti-prunin. Nuclei were then incubated in Fig. 2. Coomassie-stained SDS-PAGE gel of putative lamin appropriate secondary antibody (goat anti-mouse IgG conjugated proteins from rat liver (lanes 1-4) and pea leaf (lanes 6-9) nuclei. to FITC (Sigma) (diluted 1:100), sheep anti-rabbit IgG conjugated Lanes (1+6) total protein from the nuclear fraction. Lanes (2+7) to FITC (Sigma) (diluted 1:500), goat anti-mouse IgG/IgM con- pellet after DNAase and RNAase. Lane (3+8) pellet after high salt jugated to 10 nm colloidal gold (AuroProbe EM, Janssen Life Sci- treatment. Lanes (4+9) final pellet after detergent extraction. Lane ences Products) (diluted 1:100), or goat anti-rabbit IgG conjugated 3 (5) molecular mass standards (205, 116, 97, 66, 45, 29´ 10 Mr). to 10 nm colloidal gold (Sigma) (diluted 1:100) for 1 h. The tissue Note the similar relative molecular masss of the proteins in the processed for light level immunocytochemistry was mounted in 3 final plant and rat liver pellets near the 66´ 10 Mr marker. glycerol and viewed on a Nikon fluorescence microscope. Nuclei or lamina for EM were washed 3´ in phosphate buffer, postfixed in 1% glutaraldehyde for 30 min, washed, postfixed in 0.5% (Dwyer and Blobel, 1976). The initial stages of purification osmium tetroxide, dehydrated in 70% ethanol, stained in 0.5% were assayed microscopically to ensure that the plant uranyl acetate + 1% phosphotungstic acid for 30 min, dehydrated, nuclear preparation was free of cytoplasmic contamination embedded in epon and sectioned. Sections were viewed on a before extraction of the lamin fraction (Fig. 1). The final Hitachi 100 EM. putative lamin yield from 20 mg of total protoplast protein is 1 mg (5% of total protein). Proteins from successive fractions were separated by SDS polyacrylamide gel elec- Results

Purification of plant nuclear lamins Putative plant nuclear lamins were purified from pea nuclei isolated from etiolated pea leaf cell protoplasts following the protocol developed for purification of animal lamins

Fig. 3. Western blotting with anti-intermediate filament. Pea and rat total nuclear protein fractions were solubilized in SDS sample buffer, electrophoresed by SDS-PAGE in a 7.5-15% acrylamide gradient and transferred to an Immobilon PVDF membrane (compare with Fig. 2, lanes 1+6). (A) For comparison, final pea lamin preparation stained with Coomassie blue. Note 4 plant lamin proteins (A, B1, B2 + C) and 2 low molecular weight proteins that may be histones. (B). After incubation in monoclonal anti-IF, bound proteins were visualized via color development of Fig. 1. Micrograph of isolated pea nuclei. Nomarski micrograph the alkaline phosphatase conjugated secondary antibody. of the nuclear fraction isolated from etiolated pea leaf protoplasts Lane B1: pea (note plant protein bands that react with anti-IF). before further purification of lamins. The clumped nuclei are Lane B2: rat. No other blotted proteins bind anti-IF. Arrows mark 3 relatively free of cytoplasmic contamination. Bar, 50 µm. 66 and 49.5´ 10 Mr. 410 A. K. McNulty and M. J. Saunders

Fig. 4. Western blotting with Fig. 5. Solubilization of plant nuclear lamins. Pea lamins were anti-human lamin B. Pea, rat purified and run on a 7.5-15% SDS PAGE gel. Lane 3 contains and HeLa cell nuclear protein the final supernatant and lane 4 is the final pellet. Note the fractions were solubilized in presence of 4 lamin bands (lamins A, B1, B2 and C) in lane 4 3 SDS sample buffer, migrating near the 66´ 10 Mr marker (lane 1, arrow). One half of electrophoresed by SDS-PAGE the final lamin pellet was treated with 0.02 N NaOH and in a 7.5-15% acrylamide centrifuged at 140,000 g for 15 min. The proteins solubilized by gradient and transferred to an the NaOH treatment (lamins A, B1 and C) are found in the Immobilon PVDF membrane supernatant fraction (lane 5). The NaOH treatment pellet (lane 6) and incubated in polyclonal contains one band corresponding to lamin B2 and low molecular anti-human lamin B. Bound weight proteins that could be histones. proteins were visualized via color development of the alkaline phosphatase animal intermediate filaments and lamins, we analyzed conjugated secondary antibody. them by western blots probed with a monoclonal anti-IF (A) Lane 1: BSA control. Lane antibody (obtained from hybridoma cells supplied by Amer- 2: total pea nuclear proteins. Lane 3: pea lamin fraction. ican Type Culture Collection (TIB 131)) (Pruss et al., Lane 4: total rat nuclear proteins. Lane 5: rat lamin/pore fraction. 1981). This antibody recognizes a conserved region of all Lane 6: total HeLa cell nuclear proteins. Lane 7: Hela cell classes of intermediate filaments including mammalian lamin/pore fraction. Note the major lamin B band in pea rat and nuclear lamins (Traub et al., 1988). Total nuclear prepara- HeLa cell lanes. (B) Lane 1: rat nuclear protein. Lane 2: mixture tions from pea and rat were used for blotting to include of rat plus pea nuclear proteins. Lane 3: pea nuclear proteins. Note other proteins as a control for nonspecific binding. The anti- 2 plant protein bands that react with anti-human lamin B that IF antibody binds strongly to at least 3 proteins in the pea migrate slightly faster than rat or Hela cell lamin B (Mr 64,000 + nuclear fraction (Fig. 3B: lane 1) (M 68,000-58,000) that 3 r 62,000). Arrowheads mark 80 and 49.5´ 10 Mr. migrate close to rat nuclear proteins that are also recognized by this antibody (Fig. 3B: lane 2). The pea fraction trophoresis (PAGE) using 10% acrylamide gels and visu- contains bands that stain much darker than comparable rat alized with Coomassie Blue (Fig. 2). Similar fractions were bands of the same Mr. No other pea protein bands that were prepared from rat liver nuclei for comparison. There is blotted are bound by anti-IF. These plant nuclear proteins increasing purity of plant nuclear proteins with a relative appear to share an epitope with animal intermediate fila- molecular mass similar to those of animal lamins. Two ments and therefore exhibit some homology with type V major bands are visible in the final plant pellet (lane 9) each IF. with a relative molecular mass close to the proteins in the To determine if the putative lamin proteins we had iso- rat lamin fraction (lane 4). The putative plant lamins can lated from pea nuclei were in fact related to mammalian be resolved into 4 discrete protein bands (lamins A, lamins, we probed an immunoblot of pea nuclear proteins, putative pea nuclear lamins, rat nuclear proteins, rat B1,B2,C) (Mr 68,000-58,000) using a 7.5-15% gradient gel (Fig. 3A). Minor contamination with low molecular weight lamin/pore complexes, HeLa cell nuclei and HeLa cell proteins, possibly histones, can be detected. lamin pore/complexes with an antibody made (in rabbit) against a synthetic peptide derived from the human lamin sequence that encodes the nuclear transport signal region Western blot analysis using anti-intermediate filament and of lamin B (Cance et al., 1992) (Fig. 4A). Anti-human lamin anti-human lamin B B recognizes two close protein bands in pea (lanes 2+3; To investigate the similarity of these plant proteins to Fig 4B: lane 3) that migrate slightly faster (Mr 65,000 + Plant nuclear lamins 411

63,000) than the major band evident in rat (lanes 4+5) and sequence homology with the nuclear transport signal of HeLa cells (lanes 6+7). Rat and pea lamins can be distin- human and rat lamins. Plant lamin B1 is either more abun- guished when run in the same lane (Fig. 4B). There may dant or has a higher affinity for this antibody than lamin be two isoforms of plant lamin B (B1 + B2) that share B2. There are minor bands (close to the 49,500 Mr, arrow)

Fig. 6. Immunocytochemical localization of anti-intermediate filament in pea nuclei and lamina. Detergent-extracted pea nuclei (A+C) and lamina (B) were treated with a primary antibody (A: anti-tubulin; B+C+D: anti-IF) and secondary antibody (anti-mouse IgG/IgM conjugated to 10 nm colloidal gold for EM (A+B+C) or anti-mouse IgG conjugated to FITC for light level immunofluorescence (D). No binding can be seen in control nuclei (A). However, colloidal gold deposits (arrowheads) or fluorescence indicate localization of intermediate filaments in filamentous lamina (L) and in the nuclear matrix (NM) of extracted nuclei (C+D). No binding is seen to the residual or whole nucleoli (Nu). Bar, A: 0.5 µm; B: 0.33 µm; C: 0.25 µm; D: 10 µm. 412 A. K. McNulty and M. J. Saunders evident in the rat nuclear preparation that may be break- membrane proteins. Animal lamins A + C are easily down products or isoforms of rat lamin B. extracted, while B can remain associated with the nuclear envelope (Gerace and Blobel, 1980). The proteins from the Solubility characteristics of plant lamins ±NaOH pellets and corresponding supernatant fractions To investigate the solubility characteristics of plant lamins, were prepared for electrophoresis on a 7.5-15% SDS PAGE nuclear lamins were purified as described above and treated gel (Fig. 5). Three of the plant proteins which were origi- with NaOH to determine whether they behave as extrinsic nally in the nuclear pellet (lane 4), become solubilized upon

Fig. 7. Immunocytochemical localization of anti-lamin B in pea nuclei. Detergent-extracted pea nuclei (A+B+C) were treated with a primary antibody (anti-human lamin B) (A+C) or anti-prunin (B) and secondary antibody (anti-mouse IgG/IgM conjugated to 10 nm colloidal gold for EM (B+C) or anti-mouse IgG conjugated to FITC for light level immunoﬂuorescence (A). No binding can be seen in control nuclei to nuclear matrix (NM) or nucleoli (Nu) (B). However, extensive colloidal gold deposits indicate localization of lamin B in the nuclear matrix (NM) of extracted nuclei (C). Bar, A: 10 µm; B: 0.5 µm; C: 0.1 µm. Plant nuclear lamins 413

NaOH treatment, and appear in the supernatant fraction Since nuclear lamins are proposed to represent the ances- (lane 5: lamins A, B1 and C), thus behaving as peripheral tral class of intermediate filaments, it is logical that of all proteins. There is one protein which remains in the NaOH- classes of intermediate filaments, lamins may be conserved treated pellet (lane 6) which corresponds to pea lamin B2, in plants. Previous reports of immunological detection of indicating that this protein may be more closely associated intermediate filaments or nuclear phosphoproteins in plants with the nuclear membrane and therefore more difficult to in plants (Dawson et al. 1985; Parke et al., 1987; Harper solubilize by NaOH treatment. et al., 1990; Beven et al., 1991) may in fact have described some nuclear lamin proteins. Light and electron microscopic immunocytochemistry One would imagine that plant nuclei could have a simi- Detergent-extracted pea nuclei and purified pea lamina were lar structure to other eukaryotic nuclei and similar regula- prepared as above and processed for indirect EM and light tion pathways as the nuclear envelope breaks down in mito- level immunocytochemistry using anti-IF (Fig. 6) and anti- sis. In vitro disassembly and M-phase specific phos- lamin B (Fig. 7). While no binding can be detected in con- phorylation of lamins by cdc2 kinase have been described trol cells (either anti-tubulin (Fig. 6A) or no primary anti- in animals (Peter et al., 1990). Reports of p34cdc2 in plants body used (not shown)), colloidal gold deposits (indicating (John et al., 1989; Feiler and Jacobs, 1990) lend further specific binding of anti-IF) can be seen within the matrix weight to the hypothesis that regulation of mitosis in plants of treated nuclei (Fig. 6C). There is no extranuclear stain- could include phosphorylation of nuclear lamins. Plants ing or binding to the nucleolus. The purified filamentous contain all the elements of a Ca2+-regulated signalling path- lamina also exhibits extensive binding while residual nuleoi way that is proposed to culminate in protein phosphoryla- in this fraction are not recognized by the antibody (Fig. 6B). tion during cytokinin-stimulation of cell division. Light level immunofluorescence also indicates binding Cytokinin-induced phosphorylation of proteins with relative 3 throughout the nuclear matrix but not to nucleoli (Fig. 6D) molecular mass between 55 and 65´ 10 Mr has been while control nuclei were not fluorescent (not shown). described in the moss Funaria (Saunders, 1990). Lamins A similar pattern is seen in both light (Fig. 7A) and EM may be substrates for plant kinases and phosphatases in this micrographs (Fig. 7C) using anti-lamin B. There are no col- hormonally-triggered signal transduction pathway leading loidal gold deposits in control nuclei (no primary antibody to cell division. (not shown) or anti-prunin (Fig. 7B)) but extensive gold Since the nuclear lamina is not only structurally impor- deposits or fluorescence are obvious within the nuclear tant but may be essential for control of cell division, these matrix of nuclei treated with anti-lamin B. results indicate that plants and animals may share important regulatory proteins of the cell cycle and mitosis. Sim- Discussion ilarities and differences in the biochemistry and physiolog- ical regulation of plant lamins as compared to animal Our results provide strong evidence for the presence in lamins, can indicate points of important evolutionary con- plants of nuclear lamin proteins and, by extension, nuclear servation and indicate future lines of research. Future type V intermediate filaments. These plant proteins can be research will determine (1) how related the putative lamins purified by procedures that have been developed for the iso- that we have identified are to animal lamins; (2) their dis- lation of animal lamins, share a common epitope with both tribution in other plants; (3) if they play a structural role in animal intermediate filaments and animal lamins and are nuclear envelope integrity; and (4) if they are phosphory- localized to the nuclear matrix. In addition, the putative lated as the plant nuclear envelope disintegrates during plant lamins A, B1 and C can be solubilized from the mitosis. nuclear matrix by NaOH treatment while B2 remains with the pelletable fraction. Both the putative plant lamins B1 We thank Drs Chaudhary and Blobel, The Rockefeller Uni- and B2 bind anti-human lamin B although they have a lower versity for the generous gift of antibodies and Joel Giewertowski relative molecular mass than rat and HeLa cell lamin B, for valuable discussions. This work was supported by a National used as controls. Two functionally different forms of lamin Science Foundation grant and USF Research and Creative Schol- arship to M.J.S. and a Natural Sciences and Engineering Research B have also been identified in both avian and mammalian Council, Canada, Postdoctoral Fellowship to A.K.M. cells (Lehner et al., 1986). The two forms of lamin B identified in plants may also have different functions since B1 may be more abundant and more easily solubilized than B2. References A major difference between plant and animal lamins is their localization. While animal lamins are found exclu- Aebi, U., Cohn, J., Buhle. and Gerace, L. (1986). The nuclear lamina is a sively around the inner perifery of the nuclear envelope, meshwork of intermediate-type filaments. Nature 323, 560-564. the proteins that we have identified as nuclear intermediate Beven, A., Guan, Y., Pert, J., Cooper, C. and Shaw, P. (1991). filaments and lamin B extend throughout the nuclear matrix. Monoclonal antibodies to plant nuclear matrix reveal intermediate filament-related components within the nucleus. J. Cell Sci. 98, 293-302. Since anti-lamin B does not discriminate between the two Cance, W. G., Chaudhary, N., Worman, H. J., Blobel, G. and Cordon forms of plant lamin B, we cannot determine if they are Cardo, C. (1992). Expression of the nuclear lamins in normal and differentially distributed as might be indicated by the sol- neoplastic human tissues. J. Exp. Clin. Cancer Res. (In press). ubilization data. It may be that there are addditional func- Dawson, P. J., Hulme, J. S. and Lloyd, C. W. (1985). Monoclonal tions for lamin-like proteins in plants that involve matrix antibody to intermediate filament antigen cross-reacts with higher plant cells. J. Cell Biol. 100, 1793-1798. organization, that are supplied by other filamentous proteins Dessev, G., Iovcheva-Dessev, C., Bischoff, J. R., Beach, D. and in animal cells. Goldman, R. (1991). A complex containing p34cdc2 and cyclin B 414 A. K. McNulty and M. J. Saunders

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