Journal of Cell Science 103, 407-414 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
407
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
nuclear envelope in vertebrates and invertebrates is the
nuclear lamina, an array of 1-3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly
homologous to intermediate filaments and are classified
as type V. We report the first purification, antigenic
characterization and immunocytochemical localization
of putative plant lamin proteins from pea nuclei. We
conclude that plant cells contain this ancestral class of
intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.
Introduction
attached to the membrane (Gerace and Blobel, 1980; Ottaviano and Gerace, 1986; Ward and Kirshner, 1990). The
nuclear envelope fragments into small vesicles as the chromosomes condense and the mitotic spindle forms. During
telophase, the lamins become dephosphorylated, associate
with chromatin, polymerize into a fibrous network and the
nuclear envelope re-forms around the decondensed chromatin (Gerace et al., 1984; Glass and Gerace, 1990). Thus
the nuclear lamins are hypothesized to be responsible both
for the structural integrity and organization of the nucleus
during interphase and function as a key regulator of the cell
cycle.
One to five lamins have been described from a wide variety of vertebrates and invertebrates including humans, rats,
mice, birds, frogs, clams and fruit flies (see Krohne and
Benavente, 1986; Osman et al., 1990). There has been some
speculation and initial evidence about the presence of intermediate filaments, lamin-type proteins and cell cycle-regulated phosphorylation events in plants. However, to the best
of our knowledge, nuclear lamin proteins have never been
isolated and described as such in plants. One would imagine that a similar nuclear structure and regulation would
exist in plant cells to those in other eukaryotic organisms.
It has been proposed that nuclear lamins are the earliest
type of intermediate filaments to have evolved (Osman et
al., 1990) and therefore it may be, that of all the types of
intermediate filaments, the nuclear lamins will be found in
plants.
Antigenically-determined intermediate filaments that are
related to type III IF (Dawson et al., 1985; Goodbody et
al., 1989; Hargreaves et al., 1989) and type II IF (Ross et
al., 1991) have been reported in the cytoplasm of higher
The nuclear envelope of eukaryotic cells is responsible for
segregating the nucleoplasm from the cytoplasm, regulating transport through nuclear pores, maintaining chromatin
organization in interphase, and disassembling and reassembling during mitosis. A major structural component of the
inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of one to three principle extrinsic membrane proteins, lamins A, B and C (Mr
70,000-60,000), which together form a fibrous network (see
Abei et al., 1986; Gerace, 1986).
Lamins are highly homologous in sequence and structure
to intermediate filament (IF) proteins (Fisher et al., 1986)
and have recently been classified as type V IF. Biochemical and molecular analyses have indicated that lamins A
and C are more closely related to each other than to lamin
B (Krohne and Benavente, 1986; McKeon et al., 1986;
Osborn and Weber, 1986). Structurally, all IF proteins share
a common central α-helical rod domain with long heptad
repeat patterns of hydrophobic residues. At the amino and
carboxyl terminal ends of the rod domains, all IF classes
share two highly conserved sequences. The rod portion is
flanked by variable, non-α-helical head and tail domains
(Geisler and Weber, 1982; Steinert et al., 1985; McKeon et
al., 1986; Fisher et al., 1986).
During interphase, lamin B binds to a receptor on the
inner nuclear envelope and lamins A and C bind to both
lamin B and decondensed chromatin. During prophase of
mitosis, it is hypothesized that a complex cascade of phosphorylation events culminates in phosphorylation of the
lamins, releasing lamins A and C, while lamin B remains
Key words: nuclear lamins, intermediate filaments, Pisum sativum
(pea).
408
A. K. McNulty and M. J. Saunders
plant cells. Monoclonal antibodies to plant nuclear matrix
proteins recognize several nuclear proteins that are
immunologically related to intermediate filaments (Beven
et al., 1991). Monoclonal antibodies to a high-salt Triton
X-100 insoluble fraction (which is characteristic of IF) from
Chlamydomonas produces both a diffuse cytoplasmic fluorescence as well as a perinuclear fluorescence in onion root
tips (Parke et al., 1987).
Mitosis promoting factor (MPF), with its associated
p34cdc2 kinase activity, has been shown to phosphorylate
lamins (Dessev et al., 1991) and purified p34cdc2 induces
lamina disassembly in isolated nuclei (Peter et al., 1990).
This protein has been immunologically detected in both
higher and lower plants including Arabidopsis, oats,
Chlamydomonas and brown and red algae (John et al.,
1989). In addition, p34 protein kinase and a cdc2 gene
homologue have been described in pea (Feiler and Jacobs,
1990).
In this paper we present biochemical and immunological
evidence for the presence and localization of 4 lamin proteins in Pisum sativum (pea) nuclei. These are antigenically
similar to both animal lamins and intermediate filaments,
and can be solubilized from the nuclear envelope by NaOH
treatment. However they appear to be distributed throught
the nuclear matrix and not restricted to the nuclear envelope.
buffer B. The pellet was assayed for purity by microscopic analysis.
To isolate the nuclear lamina, the purified nuclei were resuspended in lamina isolation buffer (lysis buffer B minus 2-mercaptoethanol and spermine) and passed through a 25-gauge needle
3 times. Nuclei were then incubated for 1 h with 250 µg/ml
DNAase I and 250 µg/ml RNAase I. Samples were then mixed at
0oC with an equal volume of high salt buffer (HSB; 20 mM TrisHCl, pH 8, 4 M NaCl, 2 mM EDTA and 2 mM DTT) and layered over a cushion of 15% sucrose in HSB. The nuclear lamina
was pelleted at 20,000 g for 30 min in a Beckman J-21 centrifuge.
The lamina pellet was washed once with 5 mM Tris-HCl, pH 7.2.
Solubilization of plant nuclear lamins
Lamins were purified from pea nuclei as described above. One
half of the final lamin pellet was treated with 0.02 M NaOH and
centrifuged at 140,000 g for 15 min. The initial and final pellets
and supernatants were run on a 7.5-15% SDS PAGE gel as
described below.
Protein electrophoresis
Protein was assayed by a modified Lowry procedure (Markwell
et al., 1978). Lamina pellets were resuspended in 1× Laemmli
sample buffer (Laemmli, 1970) and boiled for 3 min. The supernatants were loaded onto either 10% or 7.5 to 15% acrylamide
gradient SDS-PAGE gels on an equal protein basis. Gels were
electrophoresed at 40 mA constant current.
Immunoblotting and immunostaining
Materials and methods
Plant material and growth conditions
Pea (Pisum sativum cv. milk and honey) seeds were sown in vermiculite and grown in the dark at 22oC for 2 weeks. At this point
etiolated leaves were harvested for lamina isolation. In addition,
pea seeds were germinated on damp paper towels for 4 days and
the root tip excised for immunocytochemistry.
Rat and Hela cell lamina isolation
Rat liver lamins were isolated according to Dwyer and Blobel
(1976). In addition, rat and HeLa cell lamin/nuclear pore extracts
were provided by Dr. Chaudhary, The Rockefeller University.
Pea protoplast, nuclei and lamina isolation
Protoplasts were isolated from 3-4 g of etiolated pea leaves by
incubation for 8 h at room temperature in protoplast isolation
buffer (0.6 M sucrose, 0.07 g/100 ml cellulase, 0.06 g/100 ml
drieslase, 0.05 g/100 ml macerace, 0.07 g/100 ml spermine, 10
mM 2-[N-morpholino]ethanesulfonic acid, pH 5.3, 0.04% v/v 2mercaptoethanol, 10 mM NaCl and 10 mM KCl. This solution
was then filtered through a 100 µm wire mesh (Newark Wire Cloth
Company).
Protoplasts were pelleted at 1000 rev./min for 10 min in an IEC
HN-SII centrifuge fitted with an IEC model 215 swinging bucket
rotor (International Equipment Company). The pellet, containing
the protoplasts, was resuspended in 3 ml of lysis buffer (0.25 M
sucrose, 5 mM Tris-HCl, pH 7.2, 0.75 mM MgCl2, 0.5% Triton
X, 0.1 mM spermine, 0.04% v/v 2-mercaptoethanol, 2.5 mM
EDTA and 2.5 mM DTT) and sedimented for 10 min at 1000
rev./min. Protoplast lysates were layered over an equal volume of
lysis buffer (0.4 M sucrose, 5 mM Tris-HCl, pH 7.2, 0.35 mM
MgCl2, 0.04% v/v 2-mercaptoethanol, 2.5 mM EDTA, 0.1 mM
spermine and 2.5 mM DTT) and then pelleted at 1800 rev./min
for 10 min. The pellet (containing nuclei) was then resuspended
in lysis buffer (minus Triton X) and resedimented through lysis
For immunoblotting, the protein was electrophoretically transferred to IMMOBILON PVDF membranes (Millipore) from acrylamide gels at 100 V for 1 h. Blots were then either stained with
0.1% Coomassie Brilliant Blue or immunostained.
For immunostaining, blots were blocked overnight at 4oC with
10% horse serum in Tris-buffered saline + Tween 20 (TBST
buffer; 10 mM Tris-HCl, pH 9.5, 150 mM NaCl, 0.05% Tween
20). After blocking, the blots were incubated with either anti-intermediate filament (anti-IF) antibody in TBST + 1% horse serum
or with anti-human lamin B signal sequence antibody in Trisbuffered saline (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.02%
sodium azide) + 1% horse serum for 1.5 h at room temperature.
Anti-IF was diluted 1:50 and anti-human lamin B was diluted
1:800. Secondary antibodies (diluted 1:4000) were either goat antimouse IgG conjugated to alkaline phosphatase (for anti-IF) or goat
anti-rabbit IgG conjugated to alkaline phosphatase (for anti-human
lamin B), and were applied for 1 h. Color was developed by incubation of the blot in 3.3 mg nitroblue tetrazolium and 1.7 mg 5bromo-4-chloro-3-indolyl phosphate in 10 ml of alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM
MgCl2).
Controls were run with bovine serum albumin (BSA), rat liver
and HeLa cell lamin/pore complex extracts (prepared as described
above and gift of Dr. Chaudhary, Rockefeller University).
Antibody preparation
Mouse-mouse hybridoma cells that secrete a monoclonal IgG1
antibody that reacts with all classes of intermediate filaments
(American Type Culture Collection TIB 131) were cultured (Pruss
et al., 1981). The supernatant was used without further purification. Anti-human lamin B (made against a synthetic peptide
derived from the human lamin B signal sequence) polyclonal IgG
antibody (rabbit) was a gift from Drs Chaudhary and Blobel, The
Rockefeller University. Control antibodies used were a mouse
monoclonal anti-yeast tubulin (Sigma) and a rabbit polyclonal
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 permeabilized 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 sequentially 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
appropriate secondary antibody (goat anti-mouse IgG conjugated
to FITC (Sigma) (diluted 1:100), sheep anti-rabbit IgG conjugated
to FITC (Sigma) (diluted 1:500), goat anti-mouse IgG/IgM conjugated to 10 nm colloidal gold (AuroProbe EM, Janssen Life Sciences Products) (diluted 1:100), or goat anti-rabbit IgG conjugated
to 10 nm colloidal gold (Sigma) (diluted 1:100) for 1 h. The tissue
processed for light level immunocytochemistry was mounted in
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%
osmium tetroxide, dehydrated in 70% ethanol, stained in 0.5%
uranyl acetate + 1% phosphotungstic acid for 30 min, dehydrated,
embedded in epon and sectioned. Sections were viewed on a
Hitachi 100 EM.
Fig. 2. Coomassie-stained SDS-PAGE gel of putative lamin
proteins from rat liver (lanes 1-4) and pea leaf (lanes 6-9) nuclei.
Lanes (1+6) total protein from the nuclear fraction. Lanes (2+7)
pellet after DNAase and RNAase. Lane (3+8) pellet after high salt
treatment. Lanes (4+9) final pellet after detergent extraction. Lane
(5) molecular mass standards (205, 116, 97, 66, 45, 29×103Mr).
Note the similar relative molecular masss of the proteins in the
final plant and rat liver pellets near the 66×103 Mr marker.
(Dwyer and Blobel, 1976). The initial stages of purification
were assayed microscopically to ensure that the plant
nuclear preparation was free of cytoplasmic contamination
before extraction of the lamin fraction (Fig. 1). The final
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. 1. Micrograph of isolated pea nuclei. Nomarski micrograph
of the nuclear fraction isolated from etiolated pea leaf protoplasts
before further purification of lamins. The clumped nuclei are
relatively free of cytoplasmic contamination. Bar, 50 µm.
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
the alkaline phosphatase conjugated secondary antibody.
Lane B1: pea (note plant protein bands that react with anti-IF).
Lane B2: rat. No other blotted proteins bind anti-IF. Arrows mark
66 and 49.5×103 Mr.
410
A. K. McNulty and M. J. Saunders
Fig. 4. Western blotting with
anti-human lamin B. Pea, rat
and HeLa cell 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
and incubated in polyclonal
anti-human lamin B. Bound
proteins were visualized via
color development of the
alkaline phosphatase
conjugated secondary antibody.
(A) Lane 1: BSA control. Lane
2: total pea nuclear proteins.
Lane 3: pea lamin fraction.
Lane 4: total rat nuclear proteins. Lane 5: rat lamin/pore fraction.
Lane 6: total HeLa cell nuclear proteins. Lane 7: Hela cell
lamin/pore fraction. Note the major lamin B band in pea rat and
HeLa cell lanes. (B) Lane 1: rat nuclear protein. Lane 2: mixture
of rat plus pea nuclear proteins. Lane 3: pea nuclear proteins. Note
2 plant protein bands that react with anti-human lamin B that
migrate slightly faster than rat or Hela cell lamin B (Mr 64,000 +
62,000). Arrowheads mark 80 and 49.5×103 Mr.
trophoresis (PAGE) using 10% acrylamide gels and visualized with Coomassie Blue (Fig. 2). Similar fractions were
prepared from rat liver nuclei for comparison. There is
increasing purity of plant nuclear proteins with a relative
molecular mass similar to those of animal lamins. Two
major bands are visible in the final plant pellet (lane 9) each
with a relative molecular mass close to the proteins in the
rat lamin fraction (lane 4). The putative plant lamins can
be resolved into 4 discrete protein bands (lamins A,
B1,B2,C) ( Mr 68,000-58,000) using a 7.5-15% gradient gel
(Fig. 3A). Minor contamination with low molecular weight
proteins, possibly histones, can be detected.
Western blot analysis using anti-intermediate filament and
anti-human lamin B
To investigate the similarity of these plant proteins to
Fig. 5. Solubilization of plant nuclear lamins. Pea lamins were
purified and run on a 7.5-15% SDS PAGE gel. Lane 3 contains
the final supernatant and lane 4 is the final pellet. Note the
presence of 4 lamin bands (lamins A, B1, B2 and C) in lane 4
migrating near the 66×103 Mr marker (lane 1, arrow). One half of
the final lamin pellet was treated with 0.02 N NaOH and
centrifuged at 140,000 g for 15 min. The proteins solubilized by
the NaOH treatment (lamins A, B1 and C) are found in the
supernatant fraction (lane 5). The NaOH treatment pellet (lane 6)
contains one band corresponding to lamin B2 and low molecular
weight proteins that could be histones.
animal intermediate filaments and lamins, we analyzed
them by western blots probed with a monoclonal anti-IF
antibody (obtained from hybridoma cells supplied by American Type Culture Collection (TIB 131)) (Pruss et al.,
1981). This antibody recognizes a conserved region of all
classes of intermediate filaments including mammalian
nuclear lamins (Traub et al., 1988). Total nuclear preparations from pea and rat were used for blotting to include
other proteins as a control for nonspecific binding. The antiIF antibody binds strongly to at least 3 proteins in the pea
nuclear fraction (Fig. 3B: lane 1) (Mr 68,000-58,000) that
migrate close to rat nuclear proteins that are also recognized by this antibody (Fig. 3B: lane 2). The pea fraction
contains bands that stain much darker than comparable rat
bands of the same Mr. No other pea protein bands that were
blotted are bound by anti-IF. These plant nuclear proteins
appear to share an epitope with animal intermediate filaments and therefore exhibit some homology with type V
IF.
To determine if the putative lamin proteins we had isolated from pea nuclei were in fact related to mammalian
lamins, we probed an immunoblot of pea nuclear proteins,
putative pea nuclear lamins, rat nuclear proteins, rat
lamin/pore complexes, HeLa cell nuclei and HeLa cell
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
of lamin B (Cance et al., 1992) (Fig. 4A). Anti-human lamin
B recognizes two close protein bands in pea (lanes 2+3;
Fig 4B: lane 3) that migrate slightly faster (Mr 65,000 +
Plant nuclear lamins
63,000) than the major band evident in rat (lanes 4+5) and
HeLa cells (lanes 6+7). Rat and pea lamins can be distinguished when run in the same lane (Fig. 4B). There may
be two isoforms of plant lamin B (B1 + B2) that share
411
sequence homology with the nuclear transport signal of
human and rat lamins. Plant lamin B1 is either more abundant or has a higher affinity for this antibody than lamin
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 breakdown products or isoforms of rat lamin B.
Solubility characteristics of plant lamins
To investigate the solubility characteristics of plant lamins,
nuclear lamins were purified as described above and treated
with NaOH to determine whether they behave as extrinsic
membrane proteins. Animal lamins A + C are easily
extracted, while B can remain associated with the nuclear
envelope (Gerace and Blobel, 1980). The proteins from the
±NaOH pellets and corresponding supernatant fractions
were prepared for electrophoresis on a 7.5-15% SDS PAGE
gel (Fig. 5). Three of the plant proteins which were originally 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
NaOH treatment, and appear in the supernatant fraction
(lane 5: lamins A, B1 and C), thus behaving as peripheral
proteins. There is one protein which remains in the NaOHtreated pellet (lane 6) which corresponds to pea lamin B2,
indicating that this protein may be more closely associated
with the nuclear membrane and therefore more difficult to
solubilize by NaOH treatment.
Light and electron microscopic immunocytochemistry
Detergent-extracted pea nuclei and purified pea lamina were
prepared as above and processed for indirect EM and light
level immunocytochemistry using anti-IF (Fig. 6) and antilamin B (Fig. 7). While no binding can be detected in control cells (either anti-tubulin (Fig. 6A) or no primary antibody used (not shown)), colloidal gold deposits (indicating
specific binding of anti-IF) can be seen within the matrix
of treated nuclei (Fig. 6C). There is no extranuclear staining or binding to the nucleolus. The purified filamentous
lamina also exhibits extensive binding while residual nuleoi
in this fraction are not recognized by the antibody (Fig. 6B).
Light level immunofluorescence also indicates binding
throughout the nuclear matrix but not to nucleoli (Fig. 6D)
while control nuclei were not fluorescent (not shown).
A similar pattern is seen in both light (Fig. 7A) and EM
micrographs (Fig. 7C) using anti-lamin B. There are no colloidal gold deposits in control nuclei (no primary antibody
(not shown) or anti-prunin (Fig. 7B)) but extensive gold
deposits or fluorescence are obvious within the nuclear
matrix of nuclei treated with anti-lamin B.
Discussion
Our results provide strong evidence for the presence in
plants of nuclear lamin proteins and, by extension, nuclear
type V intermediate filaments. These plant proteins can be
purified by procedures that have been developed for the isolation of animal lamins, share a common epitope with both
animal intermediate filaments and animal lamins and are
localized to the nuclear matrix. In addition, the putative
plant lamins A, B1 and C can be solubilized from the
nuclear matrix by NaOH treatment while B2 remains with
the pelletable fraction. Both the putative plant lamins B1
and B2 bind anti-human lamin B although they have a lower
relative molecular mass than rat and HeLa cell lamin B,
used as controls. Two functionally different forms of lamin
B have also been identified in both avian and mammalian
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.
A major difference between plant and animal lamins is
their localization. While animal lamins are found exclusively around the inner perifery of the nuclear envelope,
the proteins that we have identified as nuclear intermediate
filaments and lamin B extend throughout the nuclear matrix.
Since anti-lamin B does not discriminate between the two
forms of plant lamin B, we cannot determine if they are
differentially distributed as might be indicated by the solubilization data. It may be that there are addditional functions for lamin-like proteins in plants that involve matrix
organization, that are supplied by other filamentous proteins
in animal cells.
413
Since nuclear lamins are proposed to represent the ancestral class of intermediate filaments, it is logical that of all
classes of intermediate filaments, lamins may be conserved
in plants. Previous reports of immunological detection of
intermediate filaments or nuclear phosphoproteins in plants
in plants (Dawson et al. 1985; Parke et al., 1987; Harper
et al., 1990; Beven et al., 1991) may in fact have described
some nuclear lamin proteins.
One would imagine that plant nuclei could have a similar structure to other eukaryotic nuclei and similar regulation pathways as the nuclear envelope breaks down in mitosis. In vitro disassembly and M-phase specific phosphorylation of lamins by cdc2 kinase have been described
in animals (Peter et al., 1990). Reports of p34cdc2 in plants
(John et al., 1989; Feiler and Jacobs, 1990) lend further
weight to the hypothesis that regulation of mitosis in plants
could include phosphorylation of nuclear lamins. Plants
contain all the elements of a Ca 2+-regulated signalling pathway that is proposed to culminate in protein phosphorylation during cytokinin-stimulation of cell division.
Cytokinin-induced phosphorylation of proteins with relative
molecular mass between 55 and 65×103 Mr has been
described in the moss Funaria (Saunders, 1990). Lamins
may be substrates for plant kinases and phosphatases in this
hormonally-triggered signal transduction pathway leading
to cell division.
Since the nuclear lamina is not only structurally important but may be essential for control of cell division, these
results indicate that plants and animals may share important regulatory proteins of the cell cycle and mitosis. Similarities and differences in the biochemistry and physiological regulation of plant lamins as compared to animal
lamins, can indicate points of important evolutionary conservation and indicate future lines of research. Future
research will determine (1) how related the putative lamins
that we have identified are to animal lamins; (2) their distribution in other plants; (3) if they play a structural role in
nuclear envelope integrity; and (4) if they are phosphorylated as the plant nuclear envelope disintegrates during
mitosis.
We thank Drs Chaudhary and Blobel, The Rockefeller University for the generous gift of antibodies and Joel Giewertowski
for valuable discussions. This work was supported by a National
Science Foundation grant and USF Research and Creative Scholarship to M.J.S. and a Natural Sciences and Engineering Research
Council, Canada, Postdoctoral Fellowship to A.K.M.
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(Received 6 April 1992 - Accepted 13 July 1992