Genetic analysis of the relationships between the amoebal extranuclear
spindle-organizing centre and the plasmodial intranuclear spindleorganizing centre of Physarum during conjugation
H. AKHAVAN-NIAKI, L. MIR, M. L. OUSTRIN, A. MOISAND and M. WRIGHT
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique, 205 route de
Narbonne, 31077 Toulouse Cedex, France
Summary
Physarum amoebae possess an extranuclear spindleorganizing centre (abbreviated SPOC), located in a
typical centrosome with a pair of associated centrioles while plasmodia possess an intranuclear
SPOC without centrioles. In order to ascertain
whether, during conjugation, the plasmodial SPOC is
derived from the amoebal one or is not related to it,
we have constructed amoebal strains possessing two
and three SPOCs and we have used as a genetic
marker the frequency of polycentric metaphases in
order to evaluate the number of SPOCs in the
plasmodia. The results of both symmetrical crosses,
i.e. between amoebae possessing the same number of
SPOCs, and asymmetrical crosses, i.e. between
amoebae possessing a different number of SPOCs,
show that: (1) the number of SPOCs in plasmodia is
dependent upon the number of SPOCs in either one
of the two parental amoeba; (2) in no cross does the
number of plasmodial SPOCs equal the sum of the
parental amoebal SPOCs, but it corresponds to that
of only one parent without any polarity of transmission in asymmetrical crosses. These results are
consistent with the following model: (1) plasmodial
SPOCs are derived from the amoebal ones; and (2)
one set of parental SPOCs is lost, destroyed or
inactivated in the zygote.
Introduction
centrosome with two centrioles (Wright et al. 1979; Wright
et al. 1980). Plasmodia, generally diploid, result from the
conjugation of two sexually compatible amoebae, morphologically indistinguishable. In plasmodia mitosis is intranuclear and involves an intranuclear SPOC devoid of
centrioles (Guttes et al. 1968; Goodman and Ritter, 1969;
Ryser, 1970; Sakai and Shigenaga, 1972; Tanaka, 1973).
These differences, which are not related to cell ploidy
(Wille and Steffens, 1979; Youngman et al. 1981), raise
several questions. During conjugation what is the fate of
the amoebal SPOC and how does the plasmodial one arise?
Is there any relationship between them? In order to
determine whether there is a relationship between these
two apparently distinct types of SPOCs we have followed
the fate of amoebal SPOCs during conjugation. To do this
we crossed amoebae with different numbers of SPOCs and
determined the number of SPOCs in the resulting
plasmodia. This determination was made possible by the
following previous observation: in amoebal strains differing by the number of centrioles and SPOCs, the percentage
of multipolar metaphases, i.e. the apparent number of
cells with more than two spindle poles, increases exponentially with the number of SPOCs (Mir et al. 1984). The
percentage of polycentric metaphases could therefore be
used to evaluate the number of active SPOCs and to
compare parental amoebae and plasmodia resulting from
their crosses. We show that the number of active
plasmodial SPOCs depends on the number of SPOCs in
The equal distribution of chromosomes during mitosis
implies a polar structure that can be operationally defined
as the spindle-organizing centre (abbreviated SPOC),
regardless of the great diversity of their actual organization and terminology among evolutionarily distinct
eucaryotic cells (Heath, 1980). During fertilization a
regulatory process must take place, since both parental
cells possess a SPOC and the number of SPOCs does not
increase over successive sexual generations. It has been
suggested that in echinoderms (sea urchin, starfish) and
annelids (Sabellaria) the SPOC of the zygote could
originate from the spermatoozoon, while in mammals it
could come from the ovum (Peaucellier et al. 1974; Maro,
1985; Schatten et al. 1986; Sluder et al. 1989). In each case
only one of the two parental SPOCs is functional in the
zygote. However, it is not known whether these conclusions can be extended to other eucaryotic cells, since it
has been suggested that in Saccharomyces fusion of the
two spindle plaques occurs during conjugation (Byers and
Goetsch, 1975).
Physarum could be a useful model, since it diverged
earlier in evolution than other eukaryotic cells (Baroin et
al. 1988). Two distinct types of SPOCs are present in the
two life forms of Physarum (amoeba and plasmodium). In
amoebae mitosis is characterized by the complete disruption of the nuclear envelope and the presence of a typical
Journal of Cell Science 99, 265-271 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Key words: centrosome, mitotic centre, multipolar mitosis,
myxomycete.
265
either one of the two parental amoebae, without a
genetically determined polarity. These results are in
agreement with the two following hypotheses: (1) there is
a material continuity between the amoebal and plasmodial spindle-organizing centres; (2) the spindle-organizing
centre from one parent is randomly inactivated during
conjugation (see Fig. 4A, below).
Table 1. Number of plasmodia obtained from
asymmetrical crosses
Plaamodia
spindle-organizing centres
Type of
cross
1x2
Materials and methods
Strains
Physarum polycephalum diploid amoebal strains with 2 SPOCs
([299/860] and [713/957] in Table 1) were obtained by abortive
conjugation according to the method of Youngman et al. (1981)
from haploid strains LU299 (matAl, matB3) and LU860 (matAl,
matBl), and strains CH713 (matA2, matBl) and CH957 (matA2,
matB3), respectively. In each case, a clone showing biflagellated
cells (apparent average number of anterior flagella per amoeba:
1.22 and 1.18, respectively) was reisolated twice, checked for
ploidy (expected ploidy: 2.0 and 2.3; measured ploidy: 1.7 and 2.0)
and number of SPOCs by both determining the frequency of
multipolar mitoses (apparent mitotic abnormalities: 6.5 and 8.6 %
for 397 and 286 observed mitotic figures) and the observation of
two distinct interphase SPOCs (mtocl) by electron microscopy
(Mir et al. 1983; Wright et al. 1979). Amoebal clones with three
SPOCs ([299/860]B and [713/957]B in Table 1) were obtained
from the amoebal strains possessing two SPOCs ([299/860] and
[713/957]). In each case, a clone showing a pattern of flagellation
characteristic of strains possessing three SPOCs (Mir et al. 1984;
apparent average number of anterior flagella per amoeba: 1.53
and 1.46, respectively) was reisolated twice, checked for ploidy
(expected ploidy: 2.0 and 2.3; measured ploidy: 2.3 and 2.1) and
frequency of multipolar mitosis (apparent mitotic abnormalities:
13.3 and 28.9% for 589 and 379 observed mitotic figures).
Plasmodia, obtained by mixing at 22°C for 5-7 days 106 sexually
compatible amoebae from two amoebal clones differing at their
matA locus (Kawano et al. 1987a) were transferred under a
stereomicroscope to agar medium and kept as microplasmodia in
liquid medium in order to prepare mitotically synchronous
plasmodia (Wright and Tollon, 1978).
Determination of the frequency of polycentric mitoses in
amoebae and plasmodia
The percentage of mitotic abnormalities in amoebae, determined
by phase-contrast microscopy, was compared with the values
observed in reference strains (Mir et al. 1984). A second method,
based on the observation of the mitotic spindle after immunolabelling, has been used to quantify the frequency of multipolar
metaphases in both amoebae and plasmodia (Fig. 1). Exponentially growing amoebae (106 cells) washed in 3 ml of stabilizing
medium (SM: 4 M glycerol, 10 mM MgCl2, 5 m u EGTA, 100 mil
Pipes, pH6.5) were fixed in SM containing 15% (v/v) formaldehyde, permeabilized at room temperature for 3min in SM
containing 0.5% (v/v) Triton X-100, washed in SM and finally
resuspended in 0.5 ml of 0.13 M NaCl, 3.5 mM phosphate buffer,
pH7.2. The amoebal suspension was spread on multispot slides
(10/il per spot), dried at room temperature and then kept at
-20°C until use. Synchronous plasmodia undergoing mitosis
were fixed as described (Planques et al. 1989). Immunolabelling
(Planques et al. 1989) of microtubules of the mitotic spindle
(monoclonal anti-tubulin antibody YL 1/2, Kilmartin et al. 1982)
was observed with a Zeiss microscope (X100 plan-neofluar
objective, x2 optovar and x4 camera TV adaptator) equipped
with a Nocticon camera (Lhesa LH4036). Images were processed
(Sapphire from Quantel) by integrating 200 frames and applying
histogram, stretch and zoom (X2) functions. Frequencies of
multipolar metaphases were recorded from 1330, 782, 326 and 470
metaphase figures in amoebal strains [299/860], [713/957],
[299/860]B and [713/957]B (Fig. 1A) and from 700 metaphase
figures for each plasmodium (Fig. IB). The frequency of plasmodial polycentric metaphases in the presence of more than two
SPOCs depended on the stability of the strains. The frequency of
266
H. Akhavan-Niaki et al.
Amoebal
parental strains
CH713x[299/860]
CH957X [299/860]
LU860x[713/957]
LU299x[713/957]
2x3
[299/860]x[713/957]B
1x3
CH713x[299/860]B
CH957x[299/860]B
LU860x[713/957]B
LU299x[713/957]B
3
2
5
7
9
5
3
2=17
2=25
1
7
1
2-9
10
7
1
11
2
1
5
2=15
4
9
18
multipolar metaphases in the tetraploid plasmodium obtained by
crossing two amoebae with three SPOCs varied from 20 to 43 %
over 340 days and slowly decreased thereafter. After 500 days a
value as low as 4.4% was observed, while the plasmodium
exhibited a reduced ploidy as would be expected from the specific
elimination of polyploid nuclei (Dee and Anderson, 1984;
Werenskiold et al. 1988). Thus, 15-100 days after the appearance
of the first plasmodia, the frequencies of both multipolar mitoses
(Fig. 2) and ploidy were determined in order to check the stability
of the plasmodial clones.
Determination of ploidy
Ploidy of exponentially growing amoebae was determined by
cytofluorometry (Mir et al. 1983). Ploidy of plasmodia was
determined on 105 nuclei isolated from asynchronous microplasmodial shaken cultures (Kubbies and Pierron, 1983) with an
Epics C cell sorter (Coulter Electronic) after staining with 2 mM
Hoechst 33242 (Fig. 3).
Results
Principle of the experiment
The procedure consisted of crossing amoebae possessing
one, two and three spindle-organizing centres (SPOCs) in
order to analyze the number of SPOCs in the plasmodia
obtained after conjugation. In practice two clones of
sexually compatible amoebae were mixed in order to allow
conjugation. The population of minute plasmodia resulting from the cross was plated on agar medium. Then tiny
plasmodia were reisolated individually. As all plasmodia
obtained from each cross possessed the same genotype,
they all possess the same alleles at the fus loci and could
freely fuse together (Poulter and Dee, 1968; Collins and
Haskins, 1970). Thus, some tiny plasmodia obtained in a
single cross could have fused together just after conjugation, giving chimeric plasmodia. Crosses between two
compatible amoebae can be characterized by the number
of SPOCs in each parental amoebal strain. They are said to
be symmetrical when both parental strains possess the
same number of SPOCs ( l x l , 2x2 and 3x3) and
asymmetrical when amoebal strains differ in their
number of SPOCs (1x2, 1x3 and 2x3). The number of
plasmodial SPOCs was estimated by the percentage of
multipolar metaphases (Fig. 1). A relation between this
variable and the number of SPOCs has been demonstrated
in amoebae (Mir et al. 1984) and is also valid in plasmodia
(Fig. 2). It might be expected that all mitotic figures in
both amoebae and plasmodia possessing multiple SPOCs
Fig. 1. Polycentric metaphases in amoebae and plasmodia possessing three spindle-organizing centres. Upper rows:
immunolabelled spindle microtubules. Lower rows: stained chromosomes (0.2/igml" 1 DAPI (4,6-diamino-2-phenylindole).
(A) Amoebal metaphase figures ([713/9571B) showing 2-5 apparent spindle poles (Lemoine et al. 1984). (B) Plasmodial metaphases
figures ([299/860]Bx[713/957]B), showing 2-6 apparent spindle poles. Bar, 5/an.
would be multipolar. Indeed, the percentage of multipolar
metaphases detected after immunolabelling with antitubulin antibodies increased exponentially with the
number of SPOCs in the different amoebal clones that
have been studied (Mir et al. 1984). However, the number
of poles per half-spindle is generally less than the number
of parental SPOCs. For example, in amoebae and
plasmodia possessing 3 SPOCs, the apparent number of
spindle poles varied from two to six (Fig. 1). Thus, in these
amoebal and plasmodial strains, the apparent number of
spindle poles is generally lower than the six spindle poles
that could be expected from the presence of three SPOCs.
This effect is most probably due to the reorientation of the
multiple spindle poles in order to form a pseudo-bipolar
mitotic apparatus (Ring et al. 1982; Lemoine et al. 1984;
Armas-Portela et al. 1988). Although metaphase figures
frequently show an apparent pseudo-bipolarity when
viewed by immunofluorescence or phase-contrast microscopy, all these metaphase and anaphase figures are
multipolar and possess the expected number of spindle
poles when observed in three-dimensional reconstructions
obtained by electron-microscopic observations (Mir et al.
1983). The pseudo-bipolarization process that occurs
during mitosis is quite efficient, since the percentage of
apparent multipolar mitotic figures decreases during postmetaphase stages. For example, in amoebae possessing
three SPOCs, the percentage of multipolar figures decreased from 18 % in metaphase to 1.5 % in telophase. The
pseudo-bipolarization of the mitotic apparatus occurring
during mitosis in amoebal and plasmodial strains with
two and three SPOCs accounts for their viability and
relative stability, and thus permits their use in the present
study.
Symmetrical crosses
The presence of different alleles at the matA locus in the
amoebal strains with one, two and three SPOCs permitted
four crosses between haploid amoebae possessing one
SPOC ( l x l ) , one cross between diploid amoebae with two
SPOCs (2x2) and one cross between diploid amoebae with
Spindle-organizing centres in Physarum
267
100
50
I10
3
(X
0.5
1
Number of amoebal spindle-organizing centres
l
I
1
lxl
2x2
3x3
Number of amoebal spindle-organizing centres
in the parental strains
Fig. 2. Comparison of the frequency of multipolar mitoses in
amoebae with 1, 2 and 3 spindle-organizing centres and in
plasmodia obtained by crossing these amoebae. The frequencies
of multipolar metaphases were determined by indirect
immunolabelling of the mitotic spindle both in amoebae (O)
with 1, 2 or 3 SPOCs and in plasmodia (•) obtained by
symmetrical crosses between amoebae with 1 (lxl), 2 (2x2) or
3 (3x3) SPOCs. Multipolar metaphases were recorded in ten
independent plasmodia obtained from the cross 2x2, while
twelve independent determinations have been recorded in a
plasmodium obtained from the cross of type 3x3.
three SPOCs (3x3). All plasmodia obtained from a given
cross were homogeneous for their frequency of multipolar
metaphases. Each of the four diploid plasmodia obtained
from crosses of type l x l showed a very low percentage of
multipolar metaphases varying from 0.7 to 2.8%. In
contrast, the percentage of polycentric metaphases varied
from 4.6 to 15% (x, 7.22; a, 2.42) in ten independent
tetraploid plasmodia obtained from the cross of type 2x2.
These variations were not significantly different, since
they were similar to those observed between duplicate
determinations in two of these tetraploid plasmodia (4.9
and 9.7 %; 10.4 and 15 %). In no case did the percentage of
observed multipolar metaphases in tetraploid plasmodia
obtained from crosses of type 2x2 reach the frequencies
observed in plasmodia obtained from crosses of type l x l
and 3x3 (Fig. 2). As in amoebae (Mir et al. 1984), there
was an exponential relationship between the number of
SPOCs in the parental amoebae and the frequency of
multipolar metaphases obtained from symmetrical crosses
(Fig. 2). These results suggested that the number of
SPOCs in each plasmodium was identical to the number of
268
H. Akhavan-Niaki et al.
Fluorescence intensity
Fig. 3. Ploidy of plasmodia obtained in symmetrical crosses
( l x l , 2x2, 3x3) and asymmetrical crosses (1x2). The vertical
lines correspond to haploid, diploid, triploid and tetraploid
DNA contents, respectively. The haploid apogamic
plasmodium, called plasmodium 1 and obtained from the
haploid amoebal strain Cl, was used as reference. (A and B)
Agreement between expected and observed plasmodial ploidy
levels in plasmodia obtained in the various types of crosses.
Nuclei isolated from the apogamic plasmodium Cl (plasmodium
1) were mixed with nuclei isolated from plasmodia of different
ploidy levels. The diploid plasmodium 2 was obtained from the
two haploid amoebal strains LU860 and CH713 (symmetrical
cross of type l x l ) ; The tetraploid plasmodium 3 was obtained
from the two diploid amoebal strains [299/860] and [713/957]
(symmetrical cross of type 2x2). The tetraploid plasmodium 4
was obtained from the two diploid amoebal strains [299/860]B
and [713/957]B (symmetrical cross of type 3x3). The triploid
plasmodium 5 has been obtained in the asymmetrical cross of
type 1x2 involving the haploid and the diploid amoebal strains
LU860 and [713/957], respectively. (C-M) Homogeneity of
ploidy levels and heterogeneity of the apparent number of
active SPOCs in the different plasmodia obtained from a cross
of type 1x2. The nuclei isolated from eleven triploid
independent plasmodia (plasmodia 6-16) obtained in a cross of
type 1x2 involving the haploid and the diploid amoebal strains
CH957 and [299/860], were mixed with the nuclei isolated
from the apogamic haploid plasmodial strain Cl (plasmodium
1). The numbers shown between parentheses correspond to the
apparent number of SPOCs, deduced from the frequency of
multipolar metaphases and equal to 1 or 2, while all these
plasmodia were triploid.
SPOCs in each parental amoeba. A statistical analysis of
the variations of the logarithm of the frequency of
multipolar metaphases with a confidence interval of 95 %
gave values of 0.6-3.3%, 3.0-13.8% and 16.0-47.0%
multipolar metaphases for plasmodia possessing one, two
and three SPOCs, respectively.
Asymmetrical crosses
Three types of asymmetrical crosses were performed using
compatible amoebae possessing one, two or three SPOCs:
1x2, 1x3 and 2x3 (Table 1). In contrast to the homogeneity in the number of multipolar metaphases in plasmodia
obtained from symmetrical crosses, the frequencies observed in plasmodia from asymmetrical crosses were
heterogeneous. Crosses of the type 1x2 were most likely to
be informative as they could indicate whether the
frequency of polycentric metaphases was equal to or
higher than the frequency of multipolar metaphases in the
parental amoebae. Among the 42 plasmodia studied in the
four crosses of type 1x2, the frequency of multipolar
metaphases varied from 0.7 to 10.7%, suggesting that
none of these plasmodia possessed three SPOCs. In
contrast, in each of these four crosses, the frequencies of
polycentric metaphases suggested that 40 % of the plasmodia possessed one SPOC and 60 % two SPOCs. Among
the 18 plasmodia obtained from the asymmetrical cross of
type 2x3 (Table 1), the percentage of multipolar metaphases varied from 3.3 to 47.4%. It was likely that no
plasmodium possessed one SPOC, while 60 and 40 % of the
plasmodia possessed two and three SPOCs, respectively.
Thus asymmetrical crosses of type 1x2 and 2x3 show that
the number of plasmodial SPOCs cannot be larger or
smaller than the highest and lowest number of parental
SPOCs.
Four asymmetrical crosses of type 1x3 were performed
(Table 1). The percentage of multipolar metaphases varied
from 0.9 to 46.6 %. The plasmodia phenotypically similar
to plasmodia obtained from symmetrical crosses of type
2x2, found in each of these crosses, could result from the
fusion of two young plasmodia just after conjugation and
correspond to chimeric plasmodia. The apparent polarity
observed in two of the asymmetrical crosses 1x3 (Table 1;
CH957x[299/860]B and LU299x[713/957]B) could arise
from the small number of plasmodia studied, since in the
analogous genetical crosses of type 1x2 (Table 1;
CH957X [299/860] and LU299x [713/957]) this apparent
polarity was not found. Thus no evident genetic regulatory
mechanism is involved in the transmission of the parental
SPOCs to the zygote.
Plasmodial ploidy
As expected from their genetic background, plasmodia
obtained by crossing haploid amoebae were diploid
(Fig. 3A and B, plasmodium 2), while plasmodia obtained
by crossing diploid amoebae were tetraploid (Fig. 3A and
B, plasmodia 3 and 4) but they differed in the percentage of
multipolar metaphases. Although plasmodia in all asymmetrical crosses of type 1x3 or 1x2 were heterogeneous
for their frequencies of multipolar mitoses, they exhibited
a homogeneous ploidy in agreement with the haploid and
diploid DNA contents of the parental amoebae (Fig. 3;
plasmodium 5 and plasmodia 6-16). Thus, as in amoebae
(Mir et al. 1984), the frequency of polycentric mitoses in
plasmodia was independent of the DNA content of
plasmodial nuclei. Haploid apogamic plasmodia obtained
without conjugation directly from the three different
haploid amoebal strains possessing one SPOC (strain Cl in
Fig. 3, plasmodium 1; strains CH713 and CH808)
exhibited a low percentage of polycentric metaphases (0.6,
1.1 and 1.4%, respectively), suggesting that they possessed one SPOC as did the amoebal parental strains.
Likewise, the high percentage of multipolar metaphases
(50.1%) observed in the apogamic plasmodium obtained
from a diploid amoebal clone that possessed three SPOCs
([713/957]B) suggested that this plasmodium possessed
three SPOCs. Thus the number of SPOCs in apogamic
plasmodia was identical to the number of SPOCs in their
parental amoebal clone. The ploidy and the frequency of
multipolar metaphases in plasmodia obtained from crosses
and by apogamic development show that variations of
multicentric metaphases were not related to ploidy, but to
the number of SPOCs in one of the parental amoebae.
Discussion
The fate of spindle-organizing centres (SPOCs) during
successive cell cycles and sexual processes constitutes an
enigma shrouding this organelle; this is even more the
case in Physarum, where SPOC location varies at different
stages of the life cycle. It is possible to advance the
hypothesis that amoebal and plasmodial SPOCs could
either consist of products from different genes or share
some common gene products, constituting a basic structural element. In our experiments all plasmodia resulting
from each asymmetrical cross possess the same genotype.
Thus the different number of SPOCs observed in these
plasmodia could not be determined by the nuclear
genotype (Fig. 4E). We must assume that the number of
SPOCs in plasmodia depends on the number of SPOCs in
one of the two parental amoebae and that plasmodial
SPOCs are duplicated during each cell cycle. Several
mechanisms could account for the fate of the SPOCs
during conjugation. Observations made with plasmodia
resulting from asymmetrical crosses allow one to reject
three possibilities (Fig. 4B-D). The experimental data
verify only the loss or inactivation of one set of parental
SPOCs during conjugation (Fig. 4A). This model implies
that during conjugation one of the two parental amoebae
gives its set of SPOCs to the zygote while the set of SPOCs
from the other parental amoeba is lost, destroyed or
inactivated. As either parental amoeba could act as a
SPOC donor or receiver, no genetically defined polarity
seems to be involved. Assuming that each SPOC possesses
the same strength for determining which one of the two
parental amoebae would act as a donor during conjugation, the probability of a parental amoeba donating its
SPOCs to the zygote will depend of the number of SPOCs
in each parental amoeba. Thus, in crosses of type 1x2, one
third and two thirds of the zygotes are expected to possess
one or two SPOCs, respectively. Among the 42 plasmodia
obtained from crosses of type 1 x2,17 (1.2/3) and 25 (1.8/3)
plasmodia possessing one and two SPOCs were observed,
while 14 and 28 were expected. Similarly, in the case of
crosses of type 1x3, a quarter and three quarters of the
zygotes are expected to possess one and three SPOCs,
respectively. Among the 27 plasmodia obtained from
crosses of type 1x3, nine (1.3/4) and 18 (2.7/4) plasmodia
possessing one and three SPOCs were observed while
seven and 20 were expected. Thus there is a good
agreement between theoretical and observed numbers of
plasmodia with either one of the parental set of SPOCs,
suggesting that all SPOCs of a parental amoeba are
functionally equivalent and may equally inactivate the
SPOCs of the other parental amoeba. Since myxomycetes
diverged early from the main eukaryotic evolutionary line
(Baroin et al. 1988), this simple model could reflect a
primitive mechanism.
Evidence of uniparental inheritance of several organelles such as mitochondria, chloroplasts and extrachromoSpindle-organizing centres in Physarum
269
Amoebae
Zygote
Plasmodia
D
Fig. 4. Models accounting for the fate of parental amoebal
spindle-organizing centres during conjugation. These models
are illustrated in the simplest case, i.e. a typical conjugation
process involving two wild-type amoebae, each possessing 1
SPOC (symmetrical cross of type l x l ) . The same basic models
and their various possibilities could be drawn for symmetrical
and asymmetrical crosses involving amoebae with 2 or 3
SPOCs. (•) Active SPOC in either amoebae (A-E) or
plasmodia (A, C and D). (O) Inactived amoebal parental SPOC
in the plasmodia resulting from the conjugation process (A and
E). (••) Active plasmodial SPOC resulting from the fusion of 2
amoebal SPOCs (B). (•) Active plasmodial SPOC made de novo
in the zygote nucleus and at the origin of all plasmodial
SPOCs. The nucleus is schematically shown as a hatched circle
without any indication of the nucleolus. In all cases described
in the legend, the symbol x indicates a cross between two
amoebae characterized by their number of SPOCs, while the
number following the arrow indicates the number of active
plasmodial SPOCs. (A) Loss or inactivation of one set of
parental (amoebal) SPOCs during conjugation, lxl—>1,
2x2->2, 3x3-»3, while 1 x 2 ^ 1 or 2, Ix3->1 or 3 and 2x3->2
or 3 according to the plasmodium. The experimental values
observed in plasmodia obtained from symmetrical and
asymmetrical crosses support this model and not the other
models (B-E). (B) Fusion between parental SPOCs. For
example the fusion of all amoebal SPOCs: l x l , 2x2, 3x3,
1x2, 1x3 and 2x3—>1. (C) Distribution of parental (amoebal)
SPOCs to each of the daughter nuclei during the first mitosis
of the zygote. The two nuclei arising from the nuclear division
of the zygote nucleus stay in the same cytoplasm (Holt and
Hutterman, 1981) thus lx2->(l + 2)/2, lx3-»(l+3)/2 and
2x3-»(2+3)/2). (D) Addition of parental (amoebal) SPOCs. For
example, the addition of all parental SPOCs: lxl->2, 2x2->4,
3x3->6, lx2->3, 1X3-+4 and 2x3-*5. (E) Inactivation or loss
of all parental (amoebal) SPOCs and de novo synthesis of a
new plasmodial SPOC. l x l - » l , 2x2->2, 3x3->3, Ix2-»a,
^ and 2x3—»c (a, b and c being three integers).
270
H. Akhavan-Niaki et al.
somal rDNA has been obtained in numerous eucaryotic
organisms from observations of either the transmission of
non-nuclear genes or DNA polymorphism. In contrast, our
conclusions concerning the inheritance of Physarum
SPOCs, which have been supported by the distribution of
the two unequal sets of parental SPOCs to the zygote, do
not imply the presence of non-Mendelian segregating
DNA. In Physarum the genes coding for rRNA are located
in the nucleus on hundreds of linear extrachromosomal
DNA molecules (Holt, 1980). The rDNA molecules of both
parental amoebae are present after conjugation in the
diploid plasmodia, although their proportions may vary
after continued plasmodial growth. After meiosis each
spore contains either one or other parental type. Thus,
uniparental inheritance of extrachromosomal rDNA molecules seems to take place during meiosis (Ferris et al.
1983). Finally, uniparental inheritance of both Physarum
mitochondrial DNA and spindle-organizing centres occurs
during conjugation and the diploid plasmodia contain
either of the two parental types (Kawano et al. 19876). The
uniparental inheritance of Physarum mitochondrial DNA
depends on the parental mating type matA (Kawano and
Kuroiwa, 1989), whereas, either one of the two parental
amoebae can donate its set of SPOCs to the diploid
plasmodium independently of the matA alleles.
Evidence presented in this study suggests a new model
for SPOC inheritance in Physarum with several important
implications. First, even in a single organism uniparental
inheritance can be mediated by different mechanisms,
depending on the organelle. Second, even when there are
no differentiated gametes, as is the case during a typical
fertilization process, one parental cell acts as a SPOC
donor while the SPOC of the other parental cell is
inactivated or lost in the zygote. Third, despite having
different ultrastructure and subcellular locations, the
SPOCs of Physarum amoebae and plasmodia share a
common constituent that is inherited or lost during zygote
formation. Perhaps, despite their morphological differences, the various pole structures of mitotic spindles of all
eucaryotic cells possess a common universal element.
The numerous gifts of antibody from Dr Kilmartin have been
indispensable for the present work and the contribution of M.
Roubinet for the ploidy measurements has been greatly appreciated. The numerous pertinent suggestions from Dr Beisson and
Dr Sperling have been a significant help in achieving a readable
manuscript. This work was supported by 'L'Association pour la
Recherche sur le Cancer' and *La Ligue Nationale Francaise
contre le Cancer'.
References
ABMAS-PORTELA, R., PAWBLETZ, N., ZIMMERMANN, H. P. AND GHOSH, S.
(1988). Microtubule rearrangements during mitosis in raultinucleate
cells. Cell Motil. Cytoskel. 9, 254-263.
BAROIN, A., PERASSO, R., QU, L H., BEUQEROLLB, G. AND BACHBLLBRIB,
J. P. (1988). Partial phylogeny of the unicellular eukaryotes based on
rapid sequencing of a portion of 28S ribosomal RNA. Proc. natn. Acad.
Sci. U.SA. 86, 3474-3478.
BYEHS, B. AND GOBTSCH, L. (1975). Behavior of spindles and spindle
plaques in the cell cycle and conjugation of Saccharomyces cerevisiae.
J. Bact. 124, 511-523.
COLLINS, 0. R. AND RASKINS, E. F. (1970). Evidence for polygenic control
of plasmodial fusion in Physarum polycephalum. Nature 226, 279-280.
DEE, J. AND ANDERSON, R. W. (1984). The effect of ploidy on the
stability of plasmodial heterokaryons in Physarum polycephalum. J.
gen. Microbiol. 131, 1167-1179.
FERRIS, P. J., VOGT, V. AND TRUTTT, C. L. (1983). Inheritance of
extrachromosomal rDNA in Physarum polycephalum. Molec. cell. Biol.
3, 635-642.
GOODMAN, E. M. AND RITTER, H. (1969). Plasmodial mitosis in Physarum
polycephalum. Arch. Protistenk. I l l , 161-169.
GUTTES, S., GUTTES, E. AND Eixis, R. A. (1968). Electron microscopic
study of mitosis in Physarum polycephalum. J. Ultrastruct. Res. 22,
508-529.
HEATH, I. B. (1980). Variant mitoses in lower eukaryotes: indicators of
the evolution of mitosis? Int. Rev. Cytol. 64, 1-80.
HOLT, C. E. (1980). The nuclear replication cycle in Physarum
polycephalum. In Growth and Differentiation in Physarum
polycephalum (ed. W. F. Dove and H. P. Rusch), pp. 9-63. Princeton
University PreBS, Princeton.
HOLT, C. E. AND HOTTERMAN, A. (1981). Physarum polycephalum:
Genetic Determination of Plasmodium Formation. Film B1337, Institut
ftir den Wissenschaftltilen Film, Gottingen.
KAWANO, S., ANDERSON, R. W., NANBA, T. AND KUROIWA, T. (19876).
Polymorphism and uniparental inheritance of mitochondrial DNA in
Physarum polycephalum. J. gen. Microbwl. 133, 3175-3182.
KAWANO, S. AND KUROIWA, T. (1989). Transmission pattern of
mitochondrial DNA during plasmodium formation in Physarum
polycephalum. J. gen. Microbiol. 135, 1559-1666.
KAWANO, S., KUROIWA, T. AND ANDERSON, R. W. (1987a). A third
mating-type locus in Physarum polycephalum. J. gen. Microbiol. 133,
2539-2546.
KILMARTIN, J. V., WRIOHT, B. AND MILSTEIN, C. (1982). Rat monoclonal
antitubulin antibodies derived by using a non secreting rat cell line.
J. Cell Biol. 93, 576-582.
KUBBIES, M. AND PIERHON, G. (1983). Mitotic cell cycle control in
Physarum polycephalum. Unprecedented insights via flow-cytometry.
Expl Cell Res. 149, 57-67.
LEMOINE, A., MIR, L. AND WRIGHT, M. (1984). Indirect
immunofluorescent staining of the microtubules in interphase and
mitotic amoebae of the Myxomycete Physarum polycephalum.
Protoplasma 120, 43-50.
MARO, B. (1985). Fertilization and the cytoskeleton in the mouse.
BioEssays 3, 18-21.
MIR, L., MOISAND, A. AND WRIOHT, M. (1983). Unusual amoebal strains
of the Myxomycete Physarum polycephalum possessing two proflagellar apparatuses. Protoplasma 118, 124-134.
MIR, L., WRIGHT, M. AND MOISAND, A. (1984). Variations in the number
of centrioles, the number of microtubule organizing centers 1 and the
percentage of mitotic abnormalities in Physarum polycephalum
amoebae. Protoplasma 120, 20-35.
PBAUCELLJER, G., GUERBIKR, P. AND BEHOBRAHD, J. (1974). Effects of
cytokalasin B on meiosis and development of fertilized and activated
eggs of Sabellaria alveolata L. (Polychaete Annelid). J. Embryol exp.
Morph. 31, 61-74.
isotubulin in the mitotic spindle of Physarum polycephalum.
Protoplasma 148, 120-129.
POULTER, R. T. M. AND DEE, J. (1968). Segregation factors controlling
fusion between plasmodia of the true slime mould Physarum
polycephalum. Genet. Res. 12, 71-79.
RING, D., HUBBLE, R AND KIRSCHNER, M. (1982). Mitosis in a cell with
multiple centrioles J Cell Biol. 94, 549-556.
RYSER, U. (1970). Die Ultrastruktur der Mitosekerne in den Plasmodien
von Physarum polycephalum. Z. Zellforsch. mikrosk. Anat. 110,
108-130.
SAKAI, A. AND SHIOBNAGA, M. (1972). Electron microscopy of dividing
cells. IV. Behaviour of spindle microtubules during nuclear division in
the plasmodium of the myxomycete, Physarum polycephalum.
Chromosoma 37, 101-116.
SCHATTEN, H., SCHATTEN, G., M A Z I A , D., B A L C Z O N , R. AND StMERLY, C.
(1986). Behavior of centrosomes during fertilization and cell division
in mouBe oocytes and in sea urchin eggs. Proc. natn. Acad. Sci. U.S-A.
83, 105-109.
SLUDER, G., MILLER, F. J., LEWIS, K., DAVISON, E D. AND RIEDER, C. L.
(1989). Centrosome inheritance in starfish zygotes' selective loss of the
maternal centrosome after fertilization. Devi Biol. 131, 567-579.
TANAKA, K (1973). Intranuclear microtubule organizing center in early
prophase nuclei of the plasmodium of the slime mold, Physarum
polycephalum. J. Cell Biol. 57, 220-224.
WERENSKIOLD, A. K., SCHEECKENBACK, T. AND VALET, G. (1988). Specific
nuclear elimination in polyploid plasmodia of the slime mold
Physarum polycephalum. Cytometry 9, 261-265.
WILLE, J. J. AND STEFFBNS, W. L. (1979). Fine structure of plasmodial
nuclei in the slime mold Physarum polycephalum. I Comparison of
diploid and haploid vegetative mitosis. Protoplasma 101, 165-180.
WRIGHT, M., MIR, L. AND MOISAND, A. (1980). The structure of the pro-
flagellar apparatus of the amoebae of Physarum polycephalum:
relationship to the flagellar apparatus. Protoplasma 103, 69-81.
WEIGHT, M., MOISAND, A. AND Mm, L. (1979). The structure of the
flagellar apparatus of the swarm cells of Physarum polycephalum.
Protoplasma 100, 231-250.
WRIGHT, M. AND TOLLON, Y. (1978). Heat sensitive factor necessary for
mitosis onset in Physarum polycephalum. Molec. gen. Genet. 163,
91-99.
YOUNGMAN, P. J., ANDERSON, R. W. AND HOLT, C. E. (1981). Two
multiallelic mating compatible loci separately regulate zygote
formation and zygote differentiation in the myxomycete Physarum
polycephalum Genetics 97, 513-530.
PLANQUES, V., DUCOMMUN, B., BERTRAND, M. A., TOLLON, Y. AND
WRJOHT, M. (1989). Variation of the immunolabelling of the a\-
(Received 14 January 1991 - Accepted 15 March 1991)
Spindle-organizing centres in Physarum
271