2861
Journal of Cell Science 107, 2861-2873 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Dinoflagellates have a eukaryotic nuclear matrix with lamin-like proteins and
topoisomerase II
Ana Mínguez1, Susana Franca2 and Susana Moreno Díaz de la Espina1,*
1Laboratorio de Biología Celular y Molecular Vegetal, Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006Madrid, Spain
2Laboratorio de Microbiología Experimental, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa, Portugal
*Author for correspondence
SUMMARY
Unicellular Dinoflagellates represent the only eukaryotic
Phylum lacking histones and nucleosomes. To investigate
whether Dinoflagellates do have a nuclear matrix that
would modulate the supramolecular organization of their
non-nucleosomal DNA and chromosomes, cells of the freeliving unarmored Dinoflagellate Amphidinium carterae
were encapsulated in agarose microbeads and submitted to
sequential extraction with non-ionic detergents, nucleases
and 2 M NaCl. Our results demonstrate that this species
has a residual nuclear matrix similar to that of vertebrates
and higher plants. The cytoskeleton-nuclear matrix
complex of A. carterae shows a relatively intricate polypeptide pattern. Immunoblots with different antibodies reveal
several intermediate filament types of proteins, one of
which is immunologically related to vertebrate lamins, confirming that these proteins are ancestral members of the IF
family, which is highly conserved in eukaryotes. A topoisomerase II homologue has also been identified in the
nuclear matrix, suggesting that these structures could play
a role in organizing the Dinoflagellate DNA in loop
domains. Taken together our results demonstrate that the
nuclear matrix is an early acquisition of the eukaryotic
nucleus, independent of histones and nucleosomes in such
a way that the mechanisms controlling the two levels of
organization in eukaryotic chromatin would be molecularly and evolutionarily independent.
INTRODUCTION
Phylogenetic trees based on sequence data of 5 S RNA and
snRNAs indicate that the Dinoflagellate lineage joins the tree
at the point of plant-animal divergence, which implies that the
absence of histones and nucleosomes in the eukaryotic
chromatin of Dinoflagellates would be the result of a secondary
loss by deletion of the histone gene cluster (Cavalier-Smith,
1981), and not an indication of a primitive state. Their unusual
chromatin composition perhaps resulted in the evolution of an
unusual chromosome architecture (Loeblich, 1984; SoyerGobillard and Géraud, 1992).
From a structural point of view, Dinoflagellates show a well
organized nuclear envelope and nucleolus. At present the
knowledge of the Molecular Biology of the Dinoflagellates is
very rudimentary and does not allow the establishment of a
precise taxonomic classification of this Phylum.
Therefore Dinoflagellates can be regarded as being very
close to prokaryotes and considered as a stage in the evolution
of eukaryotic chromatin, or be firmly placed within the eukaryotes, but having an alternative to nucleosomes in their
packaging of huge amounts of DNA, which have only limited
evolutionary potential. For this reason, the study of the higherorder oganization of the nucleus and DNA in Dinoflagellates
is crucial for understanding the processes of gene organization
in eukaryotes.
DNA has several forms of higher-order structure in eukaryotes, which are organized in a particular pattern that results in
Dinoflagellates are primitive unicellular algae that present
some prokaryotic characteristics in the organization of their
DNA and chromosomes, which lack histones and nucleosomes
(Herzog and Soyer, 1981, 1983; Herzog et al., 1984, SoyerGobillard and Herzog, 1985; Taylor, 1987a; Rizzo, 1987) and
contain a substantial replacement of thymidine by the rare base
hydroxymethyl-uracil (Steele and Rae, 1980). They represent
the only eukaryotic Phylum that lacks histones and nucleosomes. Their DNA compaction and chromosome architecture,
as well as other features in which they resemble the bacterial
nucleoid, have led several authors to assume a primitive status
for the Phylum, which would have emerged near the bottom of
the eukaryotic tree (see Loeblich, 1984).
However, recent results of comparisons of 24-28 S rRNA
sequences do not confirm the latter hypothesis but indicate a
rather late appearance of the Dinoflagellates, which represent a
monophyletic group with close relationships to yeast and
ciliates (Sala Rovira et al., 1991; Lenaers et al., 1991). Besides
the molecular structure of their rDNAs, they show other eukaryotic features such as a high DNA content/cell, the presence of
repeated sequences interspersed in a similar way to other
eukaryotes, and also a similar G+C content (see Herzog et al.,
1984). The molecular structure of the Dinoflagellate snRNAs
also appears to be eukaryotic (Reddy et al., 1983).
Key words: nuclear matrix, Dinoflagellate, lamin, topoisomerase II
2862 A. Mínguez, S. Franca and S. Moreno Díaz de la Espina
the expression of the proper genes in a specific tissue and the
correct cell timing (see Pienta et al. 1991; Getzenberg et al.,
1991, for reviews).
The first-order structure of DNA is the 2 nm right-handed
double helix, which consists of two antiparallel strands of
purines and pyrimidines held together by hydrogen bonds and
hydrophobic interactions. This B-form of DNA is in the majority
in the mammalian genome, but others such as the left-handed
helix of Z-DNA, or the alternative right-handed A-DNA, also
exist. The physical control of this first-order organization of
DNA appears to occur by methylation. The nucleosomal fibre
constitutes the second order of DNA organization. The nucleosomal core is an octamer of two copies of the histones H2A,
H2B, H3 and H4. The DNA winds around the nucleosome core,
approximately 200 bp around each histone octamer forming the
10 nm nucleosomal chromatin fibre. The H1 histone binds to
the nucleosomes and the chromatin fibre, contributing to a
further packing of the chromatin fibre. Nucleosome positioning,
as well as histone modifications are involved in the transcriptional regulation of genes at this level. The nucleosomal fibre
may be further folded into 30 nm filaments, which provide a
packing ratio of the DNA of approximately 35 to 50 within the
filament. Histone H1 plays a central role in forming and stabilizing these higher-order conformations. The 30 nm filaments
form topologically and functionally independent DNA loop
domains of approximately 60 kb, which are topologically
organized by certain nuclear matrix proteins in a sequence-controlled way, by association with the matrix-associated region
(MAR) elements, and represent yet another level of the structural organization of chromatin, in such a way that the chromatin
domains or loops most probably represent units of coordinate
gene expression (see Pienta et al., 1991).
The DNA of Dinoflagellates is organized into two chromosomal regions: the main body, which contains genetically
inactive DNA and plays a role in the maintenance of chromosome structure, involving cross-linking of DNA to the protein
matrix; and the peripheral diffuse region, which contains the
DNA active in transcription (Sigee, 1984). Both B- and Z-DNA
have been detected in the chromosomes of Dinoflagellates by
immunological methods (Soyer-Gobillard et al., 1990), and the
application of spreading techniques to Dinoflagellate nuclei
suggests that the DNA in them forms continuous loops (Oakley
and Dodge, 1979; Dodge, 1985).
Two-dimensional electrophoresis has demonstrated low Mr
basic proteins in the Dinoflagellate nucleus, which are less
abundant (only 10% of the DNA mass) and display significantly lower affinitiy for DNA than do eukaryotic core histones
(Vernet et al., 1990; Sala Rovira et al., 1991; Géraud et al.,
1991b). Psoralen-crosslinking reveals that only 20% of the
genome presents protected regions, and these are clustered in
10-15 kilobase pairs separated by highly unprotected longer
regions (Yen et al., 1978).
In higher eukaryotes, the nuclear matrix provides a threedimensional support for the specific organization and regulation of genes (Berezney, 1991; Getzenberg et al., 1991).
Therefore the investigation of a eukaryotic nuclear matrix in
Dinoflagellates, which would modulate the organization of
their DNA in loops corresponding to structural and functional
domains, and also of the interactions between some of the
proteins of this matrix and certain sequences of Dinoflagellate
DNA, would be very interesting.
To begin these studies, we chose an ‘evolved’ unarmored
free-living species: Amphidinium carterae (Dinophyceae,
Gymnodiniales) (Taylor, 1987b), which presents one of the
lowest DNA contents of the taxum (2.2 pg/nucleus) (Galleron
and Durrand, 1978), analogous to those of other typical eukaryotes like chicken and tomato (see Marie and Brown, 1993), and
a low chromosome number. All these features, and its easy
growth in dense cultures in the controlled growth phase, make
this species very appropriate for the analysis of the nuclear
matrix.
Although there exist several studies on the nuclear ultrastructure (Babillot, 1970; Oliveira and Huynh, 1989), cell cycle
(Matthys-Rochon, 1979) and DNA organization (Galleron and
Durrand 1978, 1979; Sigee, 1984) in this species, as far as we
know this is the first time in which the study of the nuclear
matrix of this or any other Dinoflagellate species has been
undertaken.
With this aim, living cells of A. carterae were microencapsulated in agarose and submitted to sequential extraction to
produce nuclear matrices in situ. These structures were studied
by electron microscopy and their proteins separated by SDSPAGE. The presence of two matrix proteins involved in the
organization of DNA loops in higher eukaryotes (lamins and
topoisomerase II) was investigated in the Dinoflagellate
nuclear matrix by immunological methods.
MATERIALS AND METHODS
Cells of Amphidinium carterae Hulburt, Dynophyceae, Gymnodiniales (Taylor, 1987b), collected from the coast of Portugal were
cultured in DV medium added with AM9 antibiotic mixture
(Provasoli, 1963), at 19-21˚C, and 3500 lux (from cool-white fluorescent lamps) on a daily 14:10 LD cycle. Cultures from 75 ml Erlenmeyer flasks in the exponential phase of growth were transferred to
1000 ml Erlenmeyer flasks and, after approximately 25 days, dense
cultures in early stationary growth phase were prepared for further
study.
Nuclear matrix preparation
A total of 2×108 cells from 3,600 ml cultures at late log phase and
early stationary phase were harvested by centrifugation at 1800 rpm
and encapsulated in 0.5% agarose microbeads according to Jackson
and Cook (1985). The beads were collected at low speed, washed,
extracted with the mild non-ionic detergent Triton X-100 for 55
minutes to lyse the cells, and submitted to sequential extraction with
10 mM Tris-HCl, 5 mM MgCl2, 1 mM PMSF, 1 mM DTT, pH 7.4,
containing in each case: 1st, 20 µg/ml DNase I, 30 minutes at 20˚C;
2nd, 0.25 mM MgCl2, 15 minutes at 4˚C; 3rd, 50 µg/ml DNase I and
50 µg/ml RNase A, 30 minutes at 20˚C; 4th, 2 M NaCl, 30 minutes
at 4˚C; and in some cases, 5th, 4 M urea, 30 minutes at 4˚C.
Protein analysis
After nuclear matrix preparation, the remaining pigments in the
pellets, which interfere with SDS during electrophoresis, were
extracted by sonication in light petroleum (b.p. 50-70˚C) according
to the method of Zurdo et al. (1991). The extraction was repeated
several times until the extract was colourless. After vacuum drying,
rehydration in buffer and dialysis, samples were extracted at 100˚C
for 5 minutes in sample buffer and proteins run in either 10% or 8%
acrylamide gels according to Laemmli (1970), as previously
described (Moreno Díaz de la Espina et al., 1991), in a miniprotean
II dual slab cell. Gels were stained with either Coomassie Blue or
silver.
Dinoflagellate nuclear matrix 2863
Immunoblotting
After electrophoresis, samples were transferred to nitrocellulose
membranes in a Minitransblot cell (Bio-Rad). After washing in 0.05%
Tween-20 in PBS and blocking with a 3% solution of dried non-fat
milk in distilled water, blots were incubated with the primary antibodies. Second antibodies were peroxidase labelled. The reaction was
revealed by the ECL western blotting system from Amersham as previously described (Mínguez and Moreno Díaz de la Espina, 1993).
Antibodies
The following antibodies and dilutions were used: a rabbit polyclonal
against chicken lamins (1:75) (Stick and Hausen, 1980) and two monoclonals: L74A2 (against lamin LI from Xenopus) and L34B4 (against
chicken lamin A; Mínguez and Moreno Díaz de la Espina, 1993); the
IFA, recognizing a very conserved epitope in all types of IF proteins
(undiluted) (Pruss et al., 1981), MAC 322 a monoclonal against carrot
cytoskeletons recognizing a very conserved cytokeratin 8 epitope
shared by plants and animals (Ross et al., 1991), and a polyclonal antihuman topoisomerase II (1:25) (Cambridge Research Biochemicals).
Immunofluorescence
Encapsulated cells and cytoskeleton-nuclear matrix fractions, were
fixed in 0.3% paraformaldehyde (PFA) in PBS, pH 7.0, for 1 hour at
4˚C, washed in the same buffer, spread on polylysine-coated slides,
air dried, quickly dipped in methanol-acetone at −20˚C and stored at
−20˚C.
After washing in PBS with 0.05% Tween, for 10 minutes at room
temperature, and blocking with 2% BSA in PBS containing 0.05%
Tween, the slides were incubated with the primary antibodies during
two separate periods of 18 seconds under microwave irradiation
according to Medina et al. (1994), and washed with the same buffer.
Incubation with the FITC-conjugated anti-rabbit IgG (Sigma) at a 1:50
dilution was performed for 1 hour at room temperature. After washing,
preparations were mounted with an antifading medium containing 90%
glycerol and 0.01% paraphenylenediamine in PBS. Negative controls
were performed by omitting the primary antibody or replacing it with
purified rabbit IgG. The reaction with the antibody was observed under
an epifluorescence microscope using a 450-490 nm filter.
Electron microscopy
Agarose beads containing either whole cells or cytoskeleton-nuclear
matrix complexes, were fixed in 4% PFA in PBS, pH 7.2, for 2 hours
at 4˚C, dehydrated in an ethanol series and embedded in LR White
acrylic resin as previously described (Mínguez and Moreno Díaz de
la Espina, 1993).
RESULTS
Microencapsulation of cells
To prevent morphological alterations and redistribution of the
cellular components during the preparation of nuclear matrices,
living cells of A. carterae were encapsulated in 0.5% agarose
microbeads. As observed under phase contrast, the cells of A.
carterae inside the agarose beads are well preserved compared
with those that were observed free in a buffer solution (Fig. 1A
and B). The beads show an average diameter of 400±170 µm
and usually contain abundant cells in their interiors. The process
of encapsulation does not produce structural alterations in the
cells, as observed in the light or electron microscope. In both
free and encapsulated cells, the nuclei show a distal position
within the cell and are less refringent than the cytoplasm, which
appears brownish-yellow due to the abundant cytoplasmic
pigments (not shown here). In the electron microscope, the
Fig. 1. Phase-contrast micrographs of A. carterae cells, free (A),
encapsulated (B) or extracted within the microbeads to produce in
situ nuclear matrices (C). The nuclei occupy a distal position within
the cell (arrowheads). (C) After sequential extraction the nuclear
matrix keeps the same position as the nuclei in situ (arrowhead).
Within the extracted cytoplasm the remnants of the pyrenoid are
evident (thin arrow). Bars: A and C, 10 µm; B, 100 µm.
encapsulated cells appear well preserved. The cell wall shows
no shrinkage and both nucleus and cytoplasm appear well
preserved without retractions, demonstrating a good penetration
of the fixatives through the agarose (Fig. 2A,B). The cytoplasm
of encapsulated cells is rich in abundant mitochondria, plastids,
2864 A. Mínguez, S. Franca and S. Moreno Díaz de la Espina
Fig. 2. Electron micrographs of
whole A. carterae cells fixed, either
free (A) or after encapsulation (B).
(A) Longitudinal section of a free A.
carterae cell; 2% glutaraldehyde/2%
osmium tetroxide fixation. The
cytoplasm (Cyt) is rich in abundant
mitochondria, plastids and starch
grains (st). The nucleus has a distal
position in relation to the flagellum
(fg) and displays the typical archshaped Dinoflagellate chromosomes
(Chr), a nucleolus (Nu) bound to the
nuclear envelope and a rich
fibrillogranular nucleoplasm (np).
(B) Transverse section of a
microencapsulated cell of A.
carterae; 4% paraformaldehyde
fixation. The cytoplasm appears well
preserved and is not retracted from
the cell wall (cw). It shows a big
chloroplast (Chl) typical of these
species and abundant vesicles. The
nucleus shows a good ultrastructural
preservation of its components: the
nuclear envelope, the nucleolus (Nu)
bound to it, chromosomes
(arrowheads) and the nucleoplasm,
although they show a lower contrast
due to the lack of osmium
postfixation. Bars, 1 µm.
vacuoles and starch grains. The nucleus has the typical archshaped chromosomes of Dinoflagellates, the nucleolus is bound
to the nuclear envelope, and there is a rich fibrogranular nucleoplasm (Fig. 2B).
Ultrastructural organization of the nuclear matrix
After sequential extraction to produce the nuclear matrices in
situ, the cells appear highly extracted in the light microscope
in relation to the non-extracted controls. The cytoplasm
appears almost colourless. Within it, the remnants of the
pyrenoid and the cytoskeletal network are the most evident
structures. The residual nucleus is also highly extracted and has
the same relative position within the cell as it had before the
extraction (Fig. 1C).
In the electron microscope the effectiveness of the extraction is very evident. The cytoplasm shows no membranous
components. It appears to be composed almost exclusively of
elements of the fibrogranular cytoskeleton that are connected
to the cell wall and the periphery of the nuclear matrix (Fig.
3A,B). The residual pyrenoid is also connected to the
cytoskeleton (results not shown here).
The nuclear matrix maintains a distal position within the cell
and shows a very constant structural organization, similar to
that of other eukaryotes, with a lamina, a complex network
Dinoflagellate nuclear matrix 2865
Fig. 3. Encapsulated cells of A.
carterae after sequential extraction
to produce nuclear matrices in situ.
The only structures surviving the
extraction are the nuclear matrix
(NM) and the cytoskeleton (CSK),
which is connected to both the cell
wall (cw) and the nuclear matrix
(NM) (A). Transverse section.
(B) Longitudinal section. The
nuclear matrix (NM) maintains the
distal position within the cell and
shows a similar organization to
those of the rest of the eukaryotes.
Bars, 1 µm.
forming the internal matrix, and a residual nucleolar matrix
(Fig. 3B). The zones previously occupied by the chromosomes
in the nucleus always appear as empty areas after the extraction (compare Figs 1A,C, and 2A,B with Fig. 3B).
Higher magnifications give detailed information of the ultrastructual organization of the Dinoflagellate nuclear matrix
(Figs 4 and 5A).
The lamina forms a continuous thick layer at the periphery
of the nucleus, and is tightly bound to the internal matrix on
its inner side (Fig. 4). The fixation and embedding conditions
used here are not the most adequate to display the fibrillar
network of the cytoskeleton, but in preparations of cytoskeleton-nuclear matrix (CSK-NM) complexes that are eventually
extracted with 4 M urea some fibres are occasionally visualized pervading the cytoskeletal network and making contact
with the nuclear lamina (not shown here). The lamina is a welldeveloped nuclear structure in Dinoflagellates, which is
evident even in unextracted cells of certain species as a dense
fibrillar layer at the inner face of the nuclear envelope (Fig.
5B). The internal matrix forms an intricate network of fibrillar
patches (formed by thin 5 nm fibres), corresponding to the
residual elements of the interchromosomal and perichromoso-
2866 A. Mínguez, S. Franca and S. Moreno Díaz de la Espina
Fig. 4. Nuclear matrix of A. carterae
with the three domains typical of
eukaryotic nuclear matrices: nucleolar
matrix (Num) keeping the general
morphology of the nucleolus, the
internal matrix (im) corresponding to
the remnants of the interchromosomal
nucleoplasm, and a lamina
(arrowheads) connected to the
cytoskeleton (CSK). Bar, 1 µm.
mal regions of the nucleus (Fig. 5A,B). Granules are scarce in
this internal matrix, but structures morphologically similar to
‘coiled bodies’, formed by twisted 10-15 nm threads, are
constant components of the matrices. The nucleolar matrix
presents a morphology similar to that of the unextracted
nucleolus with a fibrillar structure that is very similar to those
of plant and vertebrate nucleolar matrices (Fig. 4).
Protein composition of the cytoskeleton-nuclear
matrix complex
Whole Amphidinium cells have a heterogeneous polypeptide
pattern in 1-D SDS-PAGE gels, with many bands between 125
and 15 kDa. Their major components migrate at 120, 79, 66,
64, 60, 44 and 34 kDa. They also show abundant low molecular
mass components, below 24 kDa. The CSK-NM complexes
show less polypeptides than whole cells, being enriched in
components at 120, 79, 51, 28 and 26 kDa. Low molecular
mass components are less abundant in these preparations than
in whole cells (Fig. 6).
To investigate the functions of the Dinoflagellate nuclear
matrix in organizing DNA loops, we tested the presence of two
proteins known to be involved in the topological organization
of eukaryotic DNA: lamins and topoisomerase II.
Immunological detection of lamins
In these experiments, proteins transferred to nitrocellulose
membranes were incubated with an anti-lamin polyclonal
serum that recognized conserved epitopes of lamins present in
birds, plants and amphibia (Mínguez and Moreno Díaz de la
Espina, 1993) as well as two monoclonals against type A
(chicken lamin A) and type B (LI from Xenopus) lamins of vertebrates. As controls, antibodies recognizing very conserved
epitopes of higher eukaryote intermediate filament (IF)cytoskeletons were used: IFA, which reacts with an epitope
common to all types of IF proteins, and MAC 322, which recognizes a very conserved epitope of cytokeratin 8 shared by
higher plants and animals. MAC 322 shows no reaction with
the proteins of the CSK-NM of Amphidinium (not shown here).
The IFA recognizes three different bands. The most abundant
with a molecular mass of 66 kDa and two others at 62 and 58
kDa (Table 1). The polyclonal anti-lamin serum reacts with
bands at 58 and 50 kDa, while the two monoclonals recognize
bands at 62 and 50 kDa (Fig. 7; Table 1).
When extracted cells of A. carterae were incubated with the
anti-lamin serum and observed by indirect immunofluorescence the nuclear lamina appeared decorated, standing out
from the weak autofluorescence of the pigments of the Dinoflagellate cytoskeleton (Fig. 8A,C). When the IFA antibody is
used, both the cytoskeleton and nuclear matrix appear strongly
fluorescent, indicating the presence of abundant IF type
proteins in these structures (Fig. 8B,D). In negative controls,
in which incubation with the primary antibody was omitted,
only a weak autofluorescence was observed (Fig. 10C,F and
data not shown).
Table 1. Crossreactivity of antibodies against nuclear matrix and IF proteins with the residual proteins of Dinoflagellates
Immunoblotted proteins (kDa)
Antibody
Type
187
66
62
58
50
IFA
Chicken lamins
Xenopus L1
Chicken A
MAC 322
M
P
M
M
M
−
−
−
−
−
++
−
−
−
−
++
−
++
++
−
++
++
−
−
−
−
++
++
++
−
topo II
P
+++
−
−
−
−
M, monoclonal; P, polyclonal. Intensity of reaction, strong (+++); medium (++); weak (+); no reaction (−).
Homologous system
IF proteins
71 and 67
Xenopus L1 lamin
Chicken A lamin
Cytokeratin 8 (mammalian) and
42, 50, 55 (plants)
Nucleoplasmic human topo II 170
Dinoflagellate nuclear matrix 2867
Fig. 5. (A) Higher magnification of a portion of the nuclear matrix of A. carterae, to show in more detail the fine organization of the lamina and
internal matrix (im). The structures similar to coiled bodies (double arrowheads) form a part of the internal matrix. The 5 nm fibrils of the
internal matrix are displayed individualized at the periphery of the patches of the internal matrix network, and denoted by arrowheads.
(B) Section of a Gymnodinium splendens whole cell labelled with an anti-DNA antibody, which marks the chromosomes. Underneath the
nuclear envelope, there is a continuous dense thick well-organized lamina (arrowheads), only interrupted at the pore complexes (double small
arrows). Chromosomes (Chr) are decorated by gold particles. The nucleoplasm surrounding the chromosomes is fibrillar but also shows
abundant granules (arrow). Nu, nucleolus. Cyt, cytoplasm. Bars, 0.5 µm.
2868 A. Mínguez, S. Franca and S. Moreno Díaz de la Espina
Fig. 6. 1-D polypeptide patterns of A. carterae whole cells, and
residual structures after 10% SDS-PAGE. Lane 1, A. carterae
residual proteins. Their major bands (*) are different from those of
the whole cells. Lane 2, A. carterae whole cells with abundant low
molecular mass components. Lane 3, molecular mass standards
(kDa).
Fig. 7. Immunoblots of A. carterae residual proteins with antibodies
against IF proteins and lamins of different types, revealed by the
ECL-amplified peroxidase (Amersham). First lane, IFA; 2nd lane,
Xenopus L1 (type B); 3rd lane, chicken lamin A (type A); 4th lane,
polyclonal serum against chicken lamina. The bands at 62, 58 and 50
kDa are antigenically related to lamins (arrowheads), while the major
band at 66 kDa is not.
Immunological detection of topoisomerase II
The anti-topoisomerase II polyclonal antibody, directed
against a peptide of human topoisomerase II, recognizes a band
at 187 kDa in the NM-cytoskeleton of A. carterae, indicating
Fig. 8. Immunofluorescence staining of A. carterae residual
structures with the anti-chicken lamin serum (A) and IFA antibody
(B). Phase-contrast microscopy of the corresponding fields (C,D).
The anti-lamin serum stains the nuclear matrix (arrow), but not the
cytoskeleton (arrowheads) (A,C). IFA incubation produces a strong
fluorescence of the cytoskeleton (arrowhead) while the nuclear
matrix appears less fluorescent (arrow). The small arrows mark the
limits of the cells. Bars, 10 µm.
Fig. 9. Immunoblot staining of the residual proteins of A. carterae
separated in 8% SDS-PAGE. The anti-topoisomerase II antibody
recognizes a band at 178 kDa, which corresponds to the homologue
of the eukaryotic enzyme in this system.
that this enzyme or a related protein with common sequences
is a component of the Dinoflagellate nuclear matrix (Fig. 9;
Table 1).
Immunofluorescence detection of the antigen indicates that
the topoisomerase II labelling is confined to the nucleus in
whole cells (Fig. 10A,D), and to the nuclear matrix after
sequential extraction (Fig. 10B,E), while the negative controls
Dinoflagellate nuclear matrix 2869
Fig. 10. Immunofluorescence
staining of whole cells (A)
and residual structures (B) of
A. carterae with the antitopoisomerase II antibody.
(C) Incubation of residual
structures with normal rabbit
serum. (D,E,F) Phase-contrast
of the corresponding fields.
The anti-topoisomerase II
stains the nucleus
(arrowheads) in whole cells
(A), and the nuclear matrix
(arrowhead) in the residual
structures (B). The small
arrows mark the limits of the
cell. Incubation with nonimmune rabbit serum does not
produce nuclear fluorescence
(arrowheads). In some cells
fluorescence of the residual
pigments of the pyrenoid is
observed (C). Bars, 10 µm.
show no reaction except for some autofluorescence of the
cytoskeleton (Fig. 10C,F).
DISCUSSION
In spite of the interesting features of the nuclei of Dinoflagellates (see Spector, 1984, for a review), which is the only
Phylum in eukaryotes lacking histones and nucleosomes, and
presenting characteristics of both prokaryotes and eukaryotes
(Herzog et al., 1984; Rizzo, 1987, 1991), no studies to search
for a subnuclear structure responsible for the organization of
their components (Berezney, 1991; Getzenberg et al., 1991)
have been undertaken.
However, the investigation of a eukaryotic nuclear matrix in
the Dinoflagellates, regulating the organization of their nonnucleosomic DNA in functional and structural loop domains,
is very necessary to understand fully the evolution of the two
levels of chromatin organization in eukaryotes.
This situation is probably not due to a lack of interest, but
rather to methodological problems in the culture and fractionation of these systems. In fact, most of the procedures used for
the preparation of nuclear matrices require starting from
purified isolated nuclei in relatively large amounts. The
presence of a rigid cell wall and the high DNA content of most
Dinoflagellate species (Rizzo, 1987) make the isolation and
further handling and extraction of their nuclei very difficult.
To overcome these problems, we chose for our experiments
one species (A. carterae) that presents many advantages for
this kind of investigation. A. carterae is an unarmored
‘evolved’ Dinoflagellate (Taylor, 1987b) with a low DNA
content of 3 pg/haploid genome (Galleron and Durrand, 1978,
1979), similar to, or even lower than, that of several higher
plants from which nuclear matrices have been sucessfully
prepared (Marie and Brown, 1993; Moreno Díaz de la Espina
et al., 1991), and which grows very easily in dense cultures.
The availability of large amounts of starting material and the
use of the microencapsulation methods to prepare nuclear
matrices, avoiding the isolation of nuclei (Jackson and Cook,
1985; Jackson et al., 1988), along with the specific characteristics of these cells (lack of rigid theca and low DNA content),
has allowed us to investigate the existence of a nuclear matrix
in these systems.
The microencapsulation methods that have been successfully used for the preparation of in situ nuclear matrices from
cultured cells (Jackson et al., 1988; Lang et al., 1993) and
plasmodia (Waitz and Loidl, 1988) are shown to be very
reliable for the routine preparation of nuclear matrices from
Dinoflagellate species, which do not have a rigid theca. This
methodology not only facilitates the handling of the cells
during the extraction procedure but, what is more important,
avoids the isolation of nuclei, which leads to a poor recovery
of nuclear matrices. Phase-contrast as well as electron
microscopy controls confirm that the cells are well preserved
in the beads, and do not experience morphological distortion,
breakage or shift of their components during the preparation of
nuclear matrices. Extraction with the different solutions, as
well as nuclease digestions proved to be very effective and
homogeneous. These preparations also preserve the connections between the nuclear matrix and the cytoskeleton.
For all these reasons we think that this method constitutes
the first step in routine exploration of the Dinoflagellate nuclear
matrix that is applicable to other unarmored Dinoflagellate
species.
In spite of the big differences in nuclear organization
between Dinoflagellates and higher eukaryotes, our results
reveal that they have a eukaryotic nuclear matrix, very similar
to that of animals (Berezeney, 1984; Verheijen et al., 1988),
plants (Moreno Díaz de la Espina et al., 1991; Frederick et al.,
1992) and lower eukaryotes (Wunderlich and Herlan, 1977;
Waitz and Loidl, 1988; Cárdenas et al., 1990; Lang et al.,
1993), but with slight ultrastructural variations corresponding
2870 A. Mínguez, S. Franca and S. Moreno Díaz de la Espina
to the peculiarities of the nuclear organization in this Phylum
(Mínguez et al., unpublished data). These results demonstrate
that the nuclear matrix is a very conserved structure and represents an early acquisition of the eukaryotic nucleus, independent of that of histones and nucleosomes, in such a way
that the mechanisms controlling the two levels of organization
of the eukaryotic chromatin would be molecularly and evolutionarily independent.
Ultrastructural organization
The Dinoflagellate nuclear matrix has three well-defined morphological components, like those of the rest of the eukaryotes
studied so far: lamina, nucleolar matrix and internal matrix. The
presence of a well-developed lamina in Dinoflagellates is
evident, not only in the extracted matrices but also in the intact
nuclei of several species that present a lamina even much more
developed than is usual in higher eukaryotes. The immunological detection of lamin-related proteins in this structure confirms
that the lamina is an ancestral nuclear structure in eukaryotes
that should serve in essential housekeeping functions for the cell
(Dessev, 1992; Gerace, 1986), and thus has been very well
conserved in the eukaryotic kingdom. The detection of a lamina
in other lower eukaryotes evolutionarily close to Dinoflagellates
(Sogin, 1991) such as the Ciliates (Wunderlich and Herlan,
1977), yeast (Allen and Douglas, 1989; Georgatos et al., 1989)
and Myxomimycetes (Waitz and Loidl, 1988; Lang et al.,
1992), confirms that this structure probably arose in the
ancestral eukaryotic cell. On the other hand the identification of
proteins immunologically related to lamins in Dinoflagellates
confirms that these proteins are ancestral members of the IF
family that have been highly conserved during eukaryotic
evolution (Doring and Stick, 1990; Dodemont et al., 1990).
In higher eukaryotes the lamina provides integrity to the
nuclear envelope during interphase. During mitosis it is
involved in prophasic nuclear disassembly, as well as in
reassembly at late telophase (Benavente and Krohne, 1986;
McKeon, 1991; Dessev, 1992; Moir and Goldman, 1993). In
Dinoflagellates with permanently condensed chromosomes,
and a ‘closed’ mitosis that proceeds within an intact nuclear
envelope (Matthys-Rochon, 1979; Heath, 1980), the lamina,
besides providing support to the envelope, probably helps to
develop some special features in their unusual mitosis, such as
the anchoring of chromosomes through the kinetochores,
which move by the action of associated cytoplasmic microtubules that do not enter the nucleus (Oakley and Dodge, 1974;
Matthys-Rochon, 1979; Heath, 1980). The existence of
common nucleoplasmic domains for internal lamins and kinetochores in higher eukaryotes has already been suggested
(Moir and Goldmann, 1993).
The morphological and ultrastructural characteristics of the
residual nucleolus or nucleolar matrix in Dinoflagellates are
very similar to those of higher eukaryotes: vertebrates
(Verheijen et al., 1984; Berezney, 1984), plants (Mínguez and
Moreno Díaz de la Espina, unpublished data; Moreno Díaz de
la Espina et al., 1991), or of lower eukaryotes (Waizt and Loidl,
1988; Cárdenas et al., 1990). This result is not surprising if we
consider that the existence of a nucleolus for rRNA processing and ribosome subunit assembly is one of the universal characters of eukaryotes (Cavalier-Smith, 1981), and also constitutes one of the best-preserved eukaryotic nuclear features of
Dinoflagellates (Spector, 1984; Salamin-Michel et al, 1990;
Geraud et al., 1991a; Soyer-Gobillard and Geraud, 1992).
Along these lines, the substructure responsible for the coordination and spatial organization of all these processes should
also be preserved during evolution.
The structural organization of the internal matrix is basically
similar to that described in higher (Verheijen et al., 1988;
Moreno Díaz de la Espina et al., 1991) and lower (Wunderlich
and Herlan, 1977; Mitchelson et al., 1979; Waitz and Loild,
1988; Allen and Douglas, 1989) eukaryotes. It consists mainly
of basic 5-10 nm fibres and scarce 20-25 nm granules, and corresponds to the residual elements of the nucleoplasm in whole
cells. One characteristic of the internal matrix in Dinoflagellates is the presence in them of abundant structures morphologically similar to coiled bodies, but which share some
features with interchromatin granules (IG), like the content of
highly phosphorylated proteins, the presence in the nuclear
matrix and some common epitopes, but whose function in the
intact nuclei is not yet clearly understood (Mínguez et al.,
unpublished data).
Protein composition
Electrophoretic analysis shows that the polypeptide pattern of
the network formed by the nuclear matrix and the cytoskeleton in Dinoflagellates is complex and differs from that of the
whole cells. Residual proteins with variable molecular mass
values appear constantly enriched in these residual structures.
The immunological analysis performed in parallel in blots
and immunofluorescence staining of whole cells and residual
structures has allowed us to identify some of the components
of these residual structures.
The IFA antibody recognizes the consensus sequence at the
carboxy-terminal end of the rod domain of IF proteins, which
is a very conserved epitope in nearly all the IF-type proteins
described so far (Pruss et al., 1981). It reveals three different
IF type proteins, demonstrating that IF are ancestral proteins
much more widely expressed in lower eukaryotes than
currently thought. The IF antigens of Dinoflagellates do not
crossreact with another type known to be shared by plant and
animal cytoskeletons (Ross et al., 1991). As the phylogenetic
studies point to lamins as the most ancestral members of the
IF family of proteins (Doring and Stick, 1990; Dodemont et
al., 1990), one could expect that the bands recognized by IFA
would correspond to different lamins. But IFA immunofluorescence reveals that this is not the case, implying the existence
of cytoplasmic IFA epitopes in single cell eukaryotes.
The crossreactivity of the residual IF proteins of Dinoflagellates with different anti-lamin antibodies indicates that the
two bands at 62 and 58 kDa, recognized by antibodies against
the two types of vertebrate lamins, correspond to Dinoflagellate lamins, while the non-reactive band at 66 kDa, which is
the most abundant in these preparations, could be responsible
for the IFA immunofluorescence of the cytoskeleton. Immunofluorescence after incubation with these antibodies shows that
lamins are components of the well-developed lamina present
in Dinoflagellates.
The band at 50 kDa, which crossreacts with all the antilamins but not with IFA could also correspond to a lamin or a
related IF protein, due to the low reliability of this antibody in
detecting IF proteins in lower eukaryotes, in which the IFA
epitope is not always conserved in IF proteins, while a positive
reaction to IFA has always led to the molecular characteriza-
Dinoflagellate nuclear matrix 2871
tion of true IF proteins (Riemer et al., 1991). Interestingly, a
50 kDa lamin-like protein has also been detected in Physarum
nuclear matrices (Lang et al., 1992). The molecular mass
values of the bands that show crossreactivity with the antilamin antibodies are slightly lower than those reported for
lamins in higher eukaryotes, either animal (Lehner et al., 1986;
Krohne and Benavente, 1986) or plant (Mínguez and Moreno
Díaz de la Espina, 1993), but are close to the values reported
for Physarum lamins (Lang et al., 1992). Although further
experiments would be necessary to determine the biochemical
and structural characteristics of Dinoflagellate lamins, our
results confirm for the first time the presence of proteins
immunologically related to eukaryote lamins in the lamina of
the Dinoflagellate nucleus.
The presence in Dinoflagellates of a homologue of topoisomerase II, immunologically related to the eukaryotic enzyme
and with conserved molecular mass values, which presents a
eukaryotic DNA in its sequence organization, with large
amounts of repetitive DNA although lacking histones and
nucleosomes, like the prokaryotes (Cavalier-Smith, 1981;
Herzog et al., 1984), reinforces the idea of a eukaryotic origin
for this Phylum. The experimental procedure used to obtain the
matrices, using low temperatures and reducing conditions,
excludes the possibility of the stabilization of the topoisomerase II in the matrix by heat or disulphide bond formation
(Kaufmann and Shaper, 1991).
Besides its catalytic function, topoisomerase II also
performs a structural role in the maintenance of the attachment
of the eukaryotic DNA loop domains to the nuclear matrix
(Razin et al., 1991; Hsieh, 1992; Razin and Vassetzky, 1992).
Thus the detection of topoisomerase II in the Dinoflagellate
nuclear matrix strongly suggests that this structure is involved
not only in the described eukaryotic functions of their nuclei,
like those related to RNA synthesis and further processing
(Reddy et al., 1983; Liu et al., 1984; Franca, 1980), but also in
maintaining their unusual DNA in loops, which have been
already visualized in spreading preparations of Dinoflagellate
chromosomes (Dodge, 1985).
The presence of lamins and topoisomerase II, both involved
in the binding of MAR sequences to the nuclear matrix
(Ludérus et al., 1992; Razin et al., 1992), as well as the preliminary results of the experiments showing the association of
Drosophila MAR sequences with the Dinoflagellate matrix
(data not shown here), confirm that the Dinoflagellate nuclear
matrix plays a role in organizing the DNA in functional loops
(Bodnar, 1988), and therefore that the mechanisms controlling
the two levels of organization of eukaryotic chromatin, i.e.
nucleosomes and loops, are evolutionarily independent.
Implications for the evolutionary position of
Dinoflagellates
The transition between prokaryotes and eukaryotes is a
problem of cellular evolution involving structures with basic
properties, which can be studied in living species. The fact that
Dinoflagellates posses all 22 universal characteristics defined
for eukaryotes (Cavalier-Smith, 1981), but only a few of those
typical of prokaryotes, makes it very unlikely that the ancestor
of Dinoflagellates was a prokaryote.
The only confirmed prokaryotic features of this Phylum are
the lack of histones and nucleosomes, the arch-shaped structure
of their chromosomes, and the attachment of the dividing chro-
mosomes to membranes (the nuclear envelope in the case of
Dinoflagellates). On the contrary, they show many characteristics of eukaryotic cells. Their cytoplasmic organization is as
complex as that of any other eukaryotes (Dodge and Greuet,
1987), and they have similar meiosis.
Dinoflagellate DNA is fully eukaryotic in its sequence
organization with large amounts of repetitive DNA (Rizzo,
1987, 1991). Our results demonstrate that Dinoflagellates have
a topoisomerase II that is immunologically related to the
eukaryotic enzyme to negatively supercoil their DNA, which
could also play a role in organizing DNA loops. As this
enzyme appears attached to the Dinoflagellate nuclear matrix,
it would allow independent uncoiling of different chromatin
domains for gene expression as in eukaryotes.
Dinoflagellates also have a discrete S period of DNA
synthesis in the cell cycle and tandemly repeated rRNA
sequences (Herzog et al., 1984). The presence of snRNPs
involved in the proccessing of the 3′ end of mRNA also represents a eukaryotic feature of Dinoflagellates. Sequence data
of rRNAs, 5 S RNA and snRNAs have confirmed the eukaryotic position of Dinoflagellates (Lenaers et al., 1984; Liu et al.,
1984).
All the above findings confirm the eukaryotic origin of this
Phylum.
The fact that the most primitive extant Dinoflagellates, the
parasitic species and Oxyrrhis marina, have histones and chromosomes that lack the arched whorls of chromatin, suggests
that their absence in the free-living species is due to a
secondary loss (Loeblich, 1984). Along these lines, if the
common ancestor of Dinoflagellates lost their nucleosomes by
deletion of the clustered histone genes, which appears to be the
most probable mechanism in view of the molecular data
available (Loeblich, 1984; Cavalier-Smith, 1988), the regulatory system of loop domains (Bodnar, 1988) was able to
control gene expression in the absence of nucleosomes.
On the other hand, their unusual mechanism of closed
mitosis (shared with the parasitic Dinoflagellates and
Parabasalian flagellates, both of which have histones),
probably helped to protect their non-nucleosomal DNA against
nucleases during mitosis, avoiding shearing, and probably
allowed them to survive the chromosomal deletion that eliminated the clustered histone genes (Cavalier-Smith, 1981).
An interesting question that remains open is which
developed first in evolution: DNA loop domains or nucleosomes.
Experiments in progress on the association of conserved
MAR sequences with the proteins of the Dinoflagellate nuclear
matrix and also with this structure in situ, should definitively
clarify the functions of this nuclear matrix in the organization
and control of loop domains in the unusual Dinoflagellate
chromatin. They should also allow evaluation of the conservation of the MAR sequences and the corresponding binding
proteins during the evolution of eukaryotes.
The authors are indebted to Dr R. Stick for the generous gift of the
anti-lamin sera; Dr D. Fairbairn for the IFA and MAC 322 antibodies;
Mrs M. Carnota and Mrs N. Fonturbel for technical assistance; Mrs
V. Lafita for typing the manuscript; and Mrs B. Ligus-Walker for
revising the English. This work has been supported by the CICYT
(Spain) project PB91-0124, and by Funds of the agreement between
CSIC (Spain) and JNICT (Portugal). A. Mínguez is a recipient of a
fellowship from the MEC (Spain).
2872 A. Mínguez, S. Franca and S. Moreno Díaz de la Espina
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(Received 24 March 1994 - Accepted 16 June 1994)