J. Cell Set. 34, 225-232 (1978)
Printed in Great Britain © Company of Biologists Limited 1978
225
CHANGE OF THE NUCLEAR PORE
FREQUENCY DURING THE NUCLEAR CYCLE
OF PHYSARUM POLYCEPHALUM
JAN H. N. SCHEL, LEO C. A. STEENBERGEN, A. G. M. BEKERS
AND FRIEDRICH WANKA
Laboratory of Chemical Cytology, Faculty of Science, Toernooiveld, Nijviegen,
The Netlierlands
SUMMARY
The change of the nuclear pore frequency during the nuclear cycle of synchronous plasmodia
of Physarum polyceplialum was investigated. Counts were made on platinum-carbon replicas
of isolated nuclei. Pore numbers varied markedly at any given time of the nuclear cycle, possibly
due to the variable DNA contents of the nuclei. T h e average pore frequency per nucleus
increased from 336 at 1 h after mitosis to 770 at 50 min before the subsequent mitosis. T h e
results suggest a quantitative relation between the nuclear DNA content and pore frequency.
This is compatible with the hypothesis that pore complexes serve as attachment sites for
nuclear DNA.
INTRODUCTION
Nuclear envelopes are perforated by pores which are associated with an elaborate
annular structure (Franke, 1970; Wischnitzer, 1973). As the ultrastructural features
of this pore complex are very similar throughout eukaryotic organisms it has been
suggested that, in addition to serving as channels for the nuclear-cytoplasmic exchange
of material (Feldherr, 1965, 1972), they might have a function in some basic cellular
activity. In view of the apparent attachment of chromatin fibres, it has been proposed
that they play a role in the spatial organization of chromatin during the interphase
period (Comings & Okada, 19700,6; Maul, 1971; Schel& Wanka, 1973; Hoeijmakers,
Schel & Wanka, 1974). Such a role is supported by the observation that, when the
nuclear envelope disintegrates during mitosis and meiosis, annular structures remain
associated with the periphery of the chromosomes (Comings & Okada, 19706;
Sorsa, 1972; Maul, 1977).
Studies on the change of pore frequencies along the cell cycle of synchronized HeLa
cells have revealed a rapid increase at the beginning of the 5-phase (Maul et al.
1972). However, in yeast the main increase of the pore numbers seems to occur during
mitosis and early Gj-phase (Jordan, Severs & Williamson, 1977). These data were
calculated from pore densities on freeze-fractured faces of the nuclear envelopes and
from independently measured nuclear diameters. Their reliability, therefore, is
restricted by deviations from random distribution of the nuclear pores and by lack
of a regular geometry of the nuclear shape.
Correspondence should be sent to: Dr J. H. N. Schel, Department of Botany, Agricultural
University, Arboretumlaan 4, Wageningen, The Netherlands.
226
J. H. N. Schel, L. C. A. Steenbergen, A. G. M. Bekers and F. Wonka
We have investigated the pore frequencies during the nuclear cycle in macroplasmodia of Physarum polycephalum. Counts were made by electron-microscopical
examination of platinum-carbon replicas of isolated nuclei (Hoeijmakers et al. 1974)
which expose half of the nuclear surface.
MATERIALS AND METHODS
Physarum polycephalum, strain M3cIV, was grown as described by Daniel & Baldwin (1964)
and macroplasmodia were prepared by fusion according to Guttes & Guttes (1964). In order
to determine the different stages of the nuclear cycle, pieces of the macroplasmodia were fixed
in 96 % ethanol and phase-contrast micrographs were taken with a Zeiss Photomicroscope III.
The average duration of the nuclear cycle between metaphase 2 and metaphase 3 (M, and M3)
after fusion of the microplasmodia was 9 h.
Nuclei were isolated using the procedure of Mohberg & Rusch (1971) with minor modifications. At different times after Af, a plasmodium was rinsed in ice-cold water and subsequently
transferred into 20 ml 10 ITLM Tris-HCl buffer, pH7'i, containing 30 mM NaCl, 1 mM KC1
and 5 mM MgCl t . This suspension was centrifuged at 50 g for 0-5 min. The pellet was resuspended in 30 ml of the same buffer supplemented with o-i % Triton X-ioo, and homogenized
with 2 strokes of a loose-fitting Teflon homogenizer (Tri-R-Stir-R) at setting 3. The resulting
homogenate was filtered through 2 layers of milk filter and the filtrate was centrifuged for
1 min at 1000 g. The nuclear pellet was then resuspended in 0-5-1 ml of the above buffer
without Triton. All steps were carried out at 4 °C.
Preparation of the platinum-carbon replicas of the nuclei was done as described by
Hoeijmakers et al. (1974). The shadowing angle was routinely io° and the preparation was
rotated during the evaporation of the platinum. Electron micrographs were made using a Zeiss
EM 9S electron microscope. All pore counts were obtained from prints with a final magnification of 17000 x .
RESULTS
Replicas were prepared from nuclei isolated at different times between the second
and third mitosis after fusion of the microplasmodia. Typical micrographs as used for
the counting of the nuclear pores are shown in Fig. 1 A-C. Nuclear pores appeared as
annular projections with a diameter of about 100 nm (Hoeijmakers et al. 1974).
A central granule was frequently recognized (Fig. I F ) . The majority of the pore
complexes could be easily identified, but owing to a few ambiguous structures, slightly
different counts were obtained when duplicate prints of the same electron micrographs were examined. The difference between the most extreme values, however,
did not exceed 5 % of the total counts.
In contrast to the clustered pore distribution found in other lower eucaryotes
(Severs, Jordan & Williamson, 1976; Cole & Wynne, 1973) the Physarum nuclei
showed a completely random distribution of the pore complexes. We have determined frequencies of pore-to-pore distances and found that there was no significant
predominance of any distance. There was also no apparent minimum distance, since
contiguous and even merging pore structures were found quite frequently (Fig. 1 E).
The same distribution pattern was found in freeze-etched faces (Schel, 1977),
indicating that the observed structures were not significantly affected by the
preparation of the replicas. Our observations agree with those made by highresolution scanning electron microscopy in mouse liver cell nuclei (Kirschner, Rush &
Martin, 1977).
Change of nuclear pore frequency in Physarum
Fig. 1. Electron micrographs of nuclear replicas from different stages of the nuclear
cycle. The bar represents 1 /tm. A, M , + 6O min; B, M , + 135 min; C, Mi + 490 min;
D, nucleus with 2 nucleoli from Mt +135 min (pore number: 470); E, F, higher magnification showing merging pore-complexes (arrow) and central granuli (arrowheads).
227
228
jf. H. N. ScJiel, L.C.A.
Steenbergen, A.G.M.
Bekers and F. Wanka
8
A
4
1
1 1
1
8 -
;
c
"o
6 8 -2
4 -
nn
1
1
1
B
'-p—1
1—Tl
I
i
c
i
8
1
i
i
r-TI
1
1
-I
i—^ 1
1
n
1 1 1r-i1
.
1
1
1
-
4
1
1
15
20
1—1
1 1-1
i
25
30
35
Pore-frequency class
i
40
n
45
Fig. 2. Pore frequency distributions in nuclear replicas at different stages of the
nuclear cycle. Times after Mt are given in min: A, JVfj + 6o; B, Mt+ 135; c, Af,+ 390;
D, M, + 49O. Frequency classes are as follows: Class 15, 150 to 159 pores per replica,
class 16, 160 to 169 per replica, etc.
An elevation seen in each replica is caused by the nucleolus as can be inferred from
its size and position. It is located in the centre of the nucleus except for the late G2phase (Fig. 1 c), when it is located closer to the nuclear envelope (Guttes & Guttes,
1969). In preparations made 1 h after the second mitosis a few nuclei showed nucleolar fragments in the state of merging (not shown here), presumably the intermediate
structures reported to occur during the reconstitution of the nucleolus after mitosis
(Mohberg, 1974; Zellweger, Ryser & Braun, 1972). Occasionally 2 nucleoli were
observed in 1 replica (Fig. 1 D). Such cases were not bound to a particular stage of the
nuclear cycle. These nuclei showed roughly twice as many pores as those of the same
preparation containing just one nucleolus. They were not included in the data
provided below.
It can be assumed that each replica represents a fairly reproducible area of about
50 % of the total nuclear surface. Total nuclear pore counts were therefore obtained
by doubling the pore counts per replica. The pore frequencies determined in this way,
at any particular stage of the nuclear cycle, varied considerably between replicas in
the same preparation (Fig. 2), while the highest values were usually 50 % above the
lowest ones. These differences cannot be ascribed to either experimental errors or to
counting errors.
One hour after Mt the average pore number per nucleus was 336 (Fig. 3). The main
increase in pore number per nucleus took place during the 5-phase, which under the
culture conditions maintained in our laboratory starts at 10 min after M2 and lasts
Change of nuclear pore frequency in Physarum
229
for a further 2 h (Werry, 1973). The increase continued during the initial part of the
Ga-phase and then levelled off. The increase during the time recorded amounts to
about 125 % of the initial pore number. The total increase from mitosis to mitosis may
be still greater if one takes into account that the initial pore number after telophase
may be lower than the 336 found 1 h after metaphase.
Pore densities, calculated from the average nuclear diameters and average pore
600 -
400 -
o
Q-
200 -
_ 6 -
4 -
2 -
Time after M2, h
Fig. 3. Increase of average pore numbers and nuclear diameters during the nuclear
cycle. A, average pore numbers per nucleus; B, average nuclear diameters determined
from electron micrographs of replicas (O) and from phase-contrast micrographs of
ethanol-fixed smears ( • ) . Standard deviations are indicated by vertical bars. The
numbers of nuclear replicas examined are given in parentheses.
230
J. H. N. Schel, L. C. A. Steenbergen, A. G. M. Bekers and F. Wanka
numbers per nucleus were 5-5, 5-4, 7-0 and 7-3 per /im2 at 60, 135, 390 and 490 min
after M2, respectively. The absence of a significant change of the density distribution
during the nuclear cycle, in spite of the increase of the pore counts, is due to the fact
that the diameters of the nuclear replicas were also increasing, again mainly during the
5-phase. A similar curve for the increase of the nuclear diameters was found in
phase-contrast micrographs of ethanol-fixed smears (Fig. 3). It should be noted that
average pore densities, obtained by direct observation of limited nuclear areas in the
micrographs, due to stereometrical reasons, are 1-5 times greater than those calculated
for the surface of the nuclear sphere.
DISCUSSION
There are 2 major advantages in the use of Physarum for an investigation of nuclear
pore frequencies during the nuclear cycle. (1) The determination of pore counts is
fairly reliable because of both the random distribution of the pores, and the spherical
shape of the comparatively small nuclei. (2) The nuclei of the plasmodial form pass
with spontaneous and complete synchrony through the phases of the nuclear cycle.
If nuclear pore complexes are considered to be attachment sites for the nuclear DNA
(Comings & Okada, 19700,6; Maul, 1971; Schel & Wanka, 1973) a pore number of
750 as observed during late G2-phase could mean that the nuclear DNA contains
750 binding sites, or multiples thereof. In G2-phase the Physarum strain M3cIV
contains 1-15 pg DNA per nucleus (Mohberg, Babcock, Haugli & Rusch, 1973),
corresponding to a total length of 3-5 x 10s /tm. With 750 binding sites there should
be about 1 binding site for every 500 /im DNA. It has been suggested that origins
of replicons are attached to some nuclear structure (Dingman, 1974), possibly the
nuclear pore-complex (Wanka et al. 1977; Mullenders, Schel, Bekers, Eygensteyn
and Wanka, in preparation). As the replicon length of Physarum is 100 to 150 /on
(Schel, 1977), a pore complex should accommodate at least 4 origins. This suggestion
of multiple-binding sites for DNA occurring at each pore complex is compatible with
observations in whole-mount preparations of nuclei or nuclear ghosts showing several
threads of chromatin and DNA respectively, emerging from the annular structures
(Comings & Okada, 1970a; Schel & Wanka, 1973 ; Mullenders et al. in preparation).
A major difficulty in assigning a definite number of DNA-binding sites to each
pore results from the fact that the nuclei of Physarum plasmodia are highly mixoheteroploid (Mohberg et al. 1973) with chromosome numbers varying between 40
and 85. Thus the broad distribution of pore frequencies could be due to this variability
of the chromatin content. This assumption is supported by the finding in synchronous
plasmodia that nuclei with 2 nucleoli possess about twice as many pores as nuclei
with one nucleolus. However, so far there is no way to obtain unambiguous proof of
such a relationship.
The increase in the average pore frequency during the 5-phase appears to be the
straightforward consequence of the duplication of DNA sites which are destined to
become attached to the nuclear pore structures. However, due to the absence of a
G^-phase in Physarum, the appearance of new pores during the 5-phase may
Change of nuclear pore frequency in Physarum
231
originate from 2 different activities. (1) An assembly of pore complexes induced by the
reattachment of annular structures which remained associated with the chromosomes
during mitosis (Comings & Okada, 19706; Maul, 1977). (2) De novo formation of
complete pore complexes, possibly related to the duplication of specific chromatin sites.
The first process has been analysed in human melanoma cells by Maul (1977). It
has to be noted that the closed mitosis in Physarum does not implicate a direct transmission of the original nuclear envelope to the daughter nuclei. Rather the major
part of it disintegrates in telophase and largely new envelopes are assembled around
the merging chromosomes of the daughter nuclei (Ryser, 1970). That these daughter
envelopes begin with low pore frequencies is indicated by the present finding that,
even 1 h after metaphase, the pore frequencies are less than half those of parental
nuclei.
De novo formation could occur by a sequence of which the final steps may be
identical with the assembly process described by Maul (1977). However, recent
studies by scanning electron microscopy have revealed various structures which
could be intermediates in the formation of new pore complexes (Kirschner et al.
1977), as, for example, 2 associated pore complexes sharing subunits of the annular
structures. Similar structures are visible in our preparations. Such a duplication fits
with the hypothesis that the attachment of the DNA occurs by specific binding sites
(Wanka et al. 1977), which, after their replication, might induce the assembly of new
pore complexes in the immediate neighbourhood of the already existing ones.
Maul et al. (1972) have also observed an increase of the pore frequency after
mitosis and during the S-phase in HeLa cells. In yeast, on the other hand, the main
formation of pores seems to take place immediately before and after mitosis, while
no change of pore frequencies could be observed in the 5-phase (Jordan et al. 1977).
It is not clear at the moment whether they are caused by the procedures employed
for the cell synchronization. As mentioned in the Introduction, there are also some
uncertainties about the reliability of the procedures used by these authors for the
determination of the pore frequencies. Until these questions are solved it remains
difficult to judge the significance of the change of pore frequencies as an indicator
for the function of the nuclear pore complex.
We are grateful to Dr K. R. Mitchelson for help with the English text.
REFERENCES
COLE, G. T. & WYNNE, M. J. (1973). Nuclear pore arrangement and the structure of the Golgi
complex in Ochromonas danica (Chrysophyceae). Cytobios 8, 161-173.
COMINGS, D. E. & OKADA, T. A. (1970a). Association of chromatin fibers with the annuli of
the nuclear membrane. Expl Cell Res. 62, 293-307.
COMINGS, D. E. & OKADA, T. A. (19706). Association of nuclear membrane fragments with
metaphase and anaphase chromosomes as observed by whole mount electron microscopy.
Expl Cell Res. 63,62-68.
DANIEL, J. W. & BALDWIN, H. H. (1964). Methods of culture for plasmodial myxomycetes. In
Methods in Cell Physiology, vol. 1 (ed. D. M. Prescott), pp. 9—42. New York: Academic Press.
DINGMAN, C. W. (1974). Bidirectional chromosome replication: Some topological considerations. J. theor. Biol. 43, 187-195.
232
J. H. N. Schel, L. C. A. Steenbergen, A. G. M. Bekers and F. Wanka
C. M. (1965). The effect of the electron-opaque pore material on exchanges through
the nuclear annuli. J. Cell Biol. 25, 43-53.
FELDHERR, C. M. (1972). Structure and function of the nuclear envelope. Adv. cell molec.
Biol. 2, 273-307.
FRANKE, W. W. (1970). On the universality of the nuclear pore complex structure. Z. Zellforsdi.
mikrosk. Anat. 105, 405-429.
GUTTES, E. & GUTTES, S. (1964). Mitotic synchrony in the plasmodium of Physarum polycephalum and mitotic synchronization by coalescence of microplasmodia. In Methods in
Cell Physiology, vol. 1 (ed. D. M. Prescott), pp. 43-53. New York: Academic Press.
GUTTES, E. & GUTTES, S. (1969). Replication of the nucleolus-associated DNA during the
'Gj-phase' in Physarum polycephaluvi J. Cell Biol. 43, 229-236.
HOEIJMAKERS, J. H. J., SCHEL, J. H. N. & WANKA, F. (1974). Structure of the nuclear pore
complex in mammalian cells. Expl Cell Res. 87, 195-206.
JORDAN, E. G., SEVERS, N. J. & WILLIAMSON, D. H. (1977). Nuclear pore formation and the
cell cycle in Saccharomyces cerevisiae. Expl Cell Res. 104, 446-449.
KIRSCHNER, R. H., RUSLI, M. & MARTIN, T. E. (1977). Characterization of the nuclear envelope,
pore complexes, and dense lamina of mouse liver nuclei in high resolution scanning electron
microscopy.J'. CellBiol. 72,118-132.
MAUL, G. G. (1971). On the octagonality of the nuclear pore complex. J. CellBiol. 51, 558-563.
MAUL, G. G. (1977). Nuclear pore complexes-elimination and reconstruction during mitosis.
J. CellBiol. 74, 492-500.
FELDHERR,
MAUL, G. G., MAUL, H. M., SCOGNA, J. E., LIEBERMAN, M. W., STEIN, G. S., HSU, B. Y. &
BORUN, T. W. (1972). Time sequence of nuclear pore formation in phytohemagglutinin-
stimulated lymphocytes and in HeLa cells during the cell cycle. J. Cell Biol. 55, 433-447.
J. (1974). The nucleus of the plasmodial slime molds. In The Cell Nucleus, vol. 1
(ed. H. Busch), pp. 187-218. New York: Academic Press.
MOHBERG, J., BABCOCK, K. L., HAUGLI, F. B. & RUSCH, H. P. (1973). Nuclear DNA content
and chromosome numbers in the myxomycete Physarum polycephalum. Devi Biol. 34,
228-245.
MOHBERG, J. & RUSCH, H. P. (1971). Isolation and DNA content of nuclei of Physarum polycepltalum. Expl Cell Res. 66, 305-316.
MOHBERG,
MULLENDERS, L. H. F., SCHEL., J. H. N., BEKERS, A. G. M., EYGENSTEYN, J. & WANKA, F.
Whole-mount structure analysis and polypeptide composition of the rapidly sedimenting
DNA-protein complex isolated from nuclei of in vitro cultured bovine liver cells.
In preparation.
RYSER, U. (1970). Die Ultrastruktur der Mitosekerne in den Plasmodien von Physarum
polycepltalum. Z. Zellforsch. mikrosk. Anat. n o , 108-130.
SCHEL, J. H. N. (1977). Observations on Interphase Nuclei in Relation to DNA Replication.
Dissertation, University of Nijmegen.
SCHEL, J. H. N. & WANKA, F. (1973). Annular structures in isolated nuclei of Physarum
polycephalum. Expl Cell Res. 82, 315-318.
SEVERS, N. J., JORDAN, E. G. & WILLIAMSON, D. H. (1976). Nuclear pore absence from areas
of close association between nucleus and vacuole in synchronous yeast cultures. J. Ultrastruct. Res. 54, 374-387.
SORSA, V. (1972). Condensation of chromosomes during meiotic prophase. Hereditas 72,
215-222.
WANKA, F., MULLENDERS, L. H. F., BEKERS, A. G. M., PENNINGS, L. J., AELEN, J. M. A. &
EYGENSTEYN, J. (1977). Association of nuclear DNA with a rapidly sedimenting structure.
Biochem. biophys. Res. Commun. 74, 739—747.
P. A. T. J. (1973). DNA-syntliese in Physarum polycephalum. Dissertation, University
of Nijmegen.
WlSCHNiTZER, S. (1973). The submicroscopical morphology of the interphase nucleus. Int.
Rev. Cytol. 34, 1-48.
ZELLWEGER, A., RYSER, U. & BRAUN, R. (1972). Ribosomal genes of Physarum: Their isolation
and replication in the mitotic cycle. J. molec. Biol. 64, 681-691.
WERRY,
(Received 28 March 1978)