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Microbiology (1995), 141,2355-2365
REVIEW
ARTICLE
Plasmodium development in the myxomycete
Physarum polycephalum :genetic control and
ceIIular events
J uIiet Bailey
Tel: +44 116 252 3414. Fax: + 4 4 116 252 3378.
~
Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK
Keywords : development, Pbyarum pobcepbaium, gene expression, cellular organization
Why study Physarum 1
Pbysarum po&ephalum belongs to the myxomycetes, or
acellular slime moulds, which occur in soils throughout
the vegetated land masses of the world; little is known,
however, about their ecological role or abundance (Feest
& Madelin, 1985, 1988). A distinctive feature of these
simple eukaryotes is the presence of two vegetative phases
in the life cycle, each consisting of a single cell type uninucleate amoebae and multinucleate syncytial plasmodia. These two cell types differ in cellular organization,
behaviour and gene expression. This review focuses on
the development of amoebae into plasmodia in P.
pohcepbalzlm.
New approaches combining molecular genetics with
classical genetics and analyses of cell organization are now
being used to investigate the amoebal-plasmodia1 transition in P. po&epbalum. One long-term aim of these
investigations is to understand how changes in gene
expression bring about the gradual reorganization of
cellular structure and behaviour that occurs as amoebae
develop into plasmodia. Another aim is to understand
how these changes in gene expression are regulated by the
mating-type gene matA, which is known to be responsible
for the initiation of development in P. po&epbalzlm. It is
hoped that insights gained by investigating such questions
in this unicellular eukaryote will shed light on the
processes involved in the differentiation of cells in
multicellular organisms.
The two cell types of P. polycephalum
Amoebae
The amoebae of P. po&cepbalum are uninucleate and
haploid with a diameter of 10-20 pm. They show amoeboid movement and feed by phagocytosis on bacteria,
fungal spores and other micro-organisms. In the laboratory, amoebae are cultured on lawns of bacteria but
strains carrying mutant alleles of the axe genes are
additionally capable of growing in liquid axenic medium
0002-0128 0 1995 SGM
(Dee et a/., 1989). In adverse conditions, such as starvation, amoebae reversibly transform into resistant cysts,
but in favourable conditions they mate and develop into
plasmodia.
Interphase amoebae possess a microtubule-organizing
centre (MTOC) consisting of a pair of centrioles surrounded by amorphous material and associated with the
nucleus (Havercroft & Gull, 1983). An elaborate array of
microtubules radiates through the cytoplasm from this
MTOC. At mitosis, the MTOC duplicates and divides,
giving rise to the spindle poles; the nuclear membrane
breaks down (open mitosis) and a spindle with astral
microtubule arrays is formed (Havercroft & Gull, 1983).
Mitosis is followed by cytokinesis. Successive mitoses
with cytokinesis result in the formation of a colony of
genetically identical amoebae.
In moist conditions, amoebae transform into flagellates
which are unable to undergo mitosis or feed, and which
revert to the amoeba1 form in dry conditions. When an
amoeba transforms into a flagellate, the centrioles form
the basal bodies of the two flagella, and the cytoplasmic
microtubules are reorganized into five arrays of flagellar
microtubules (Wright et a/., 1988). The actin-based
microfilament system is also reorganized during this
transition (Pagh & Adelman, 1982; Uyeda & Furuya,
1985).
PIasmodia
The P. poEycepbalt/m plasmodium is a yellow, macroscopic
syncytium with an intricate network of veins and, in the
laboratory, this cell has been grown to a diameter of more
than 30 cm. Locomotion in this giant multinucleate cell
occurs as a result of protoplasmic streaming of the cell
contents within the veins; the direction of streaming
reverses every 30-60 s. Plasmodia phagocytose bacteria,
myxomycete amoebae and other microbes, but also secrete
enzymes to break down extracellular material, which is
then ingested by pinocytosis. Plasmodia can be grown in
the laboratory on bacteria, or axenically on agar or in
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J. B A I L E Y
Table 1. Cell-type-specific gene expression in P. polycephalum
~~
Gene product*
Namet
Expressed in
hap-p (Lavl-1)
UN
Plasmodia
Plasmodia
Plasmodia
Plasmodia
Amoebae
Amoebae
Amoebae
Plasmodia
Amoebae
Plasmodia
Plasmodia
Plasmodia
Amoebae
Myosin heavy chain P
UN
Plasmodia
Myosin 18K light chain A
UN
Amoebae
Myosin 18K light chain P
UN
Plasmodia
Fragmin A
UN
Amoebae
Fragmin P
UN
Plasmodia
haP-P
PlasminB
PlasminC
ProfilinP
ProfilinA
ABP-46
P-ABP
ActinD
/?1A-tubulin
alB-tubulin
a2B-tubulin
P2-tubulin
Myosin heavy chain A
Lavl-2
Lavl-3
Prop (Lavl-5)
P r o A (Lav3-1)
Lav3-4
Lav3-5
ardD
betA
altB(N)
altB(E)
betC
* hap-p, hydrophobic abundant protein
-
Evidence from
RNA analysis
RNA analysis
RNA analysis
RNA analysis
RNA analysis
RNA analysis
RNA analysis
RNA analysis
RNA and protein analysis
RNA and protein analysis
RNA and protein analysis
RNA, protein and antibody
Antibody staining and peptide
mapping
Antibody staining and peptide
mapping
Antibody staining and peptide
mapping
Antibody staining and peptide
mapping
Antibody staining and peptide
mapping
Antibody staining and peptide
mapping
Reference
Martel e t al. (1988)
Laroche e t al. (1989)
Girard e t al. (1990)
Binette e t al. (1990)
Binette e t al. (1990)
St Pierre e t al. (1993)
St Pierre e t al. (1993)
Adam e t al. (1991)
Reviewed in Burland et al. ( 993b)
Kohama e t al. (1986)
Kohama & Ebashi (1986)
Uyeda & Kohama (1987)
Uyeda & Kohama (1987)
Uyeda e t af. (1988)
Uyeda et al. (1988)
plasmodial ; ABP-46, 46 kDa actin-binding protein ; P-ABP, Plysartlm actin-bundling protein.
t Gene names are in italics and clone designations are in plain text; UN,un-named.
liquid shaken culture. Plasmodia are unable to transform
into flagellated cells but, in adverse conditions, can
reversibly transform into dormant sclerotia. In their
natural habitat, plasmodia are most often observed when
they move to the surface of the soil or leaf litter to
sporulate. When plasmodia starve in the light, sporangia
are formed; meiosis occurs in the spores, three of the four
meiotic products break down and the remaining haploid
spore is encased in a wall (Laane & Haugli, 1976). In
favourable conditions, spores hatch to release amoebae or
flagellates, thus completing the life-cycle.
In contrast to the situation in amoebae during interphase,
plasmodial microtubules do not radiate from an organizing centre but form a sparse network in the cytoplasm
(Salles-Passador e t al., 1991). The mitotic spindle in
plasmodia is nucleated by an intranuclear organizing
centre and the nuclear membrane remains intact throughout this ‘closed’ mitosis (Havercroft & Gull, 1983). The
nuclei within a plasmodium undergo mitosis synchronously but cytokinesis does not occur. The absence of
cytokinesis, together with fusions between plasmodia,
lead to a rapid increase in plasmodial size. In plasmodia,
actin and myosin are organized around the veins to
provide the propulsive force for the cytoplasmic streaming, and as a thin network under the cell membrane
(reviewed in Stockem & Brix, 1994).
2356
Molecular basis of the differences between
amoebae and plasmodia
The differences in cellular organization and behaviour
between amoebae and plasmodia are the result of differences in gene expression, the molecular bases of which are
beginning to be understood. Comparisons of the proteins
present in amoebae and plasmodia indicate that as many as
25 % of abundant proteins are either cell-type-specific in
expression, or show different levels of expression in the
two cell types (Larue e t a!., 1982; Turnock e t al., 1981).
For example, there are significant differences in the
proteins present in plasma membranes of the two cell
types (Pallotta e t al., 1984), as well as in their glyco- and
phospholipid composition (Murakami-Murofushi e t al.,
1987; Minowa e t a/., 1990).
A significant proportion of genes show cell-type-specific
expression; 5-10 % of the genes expressed in plasmodia
appear not to be expressed in amoebae, and a similar
proportion of genes show amoeba-specific expression
(Pallotta e t al., 1986; Sweeney e t al., 1987). In several
cases, different members of multi-gene families are expressed in amoebae and plasmodia (Table 1). For example,
amoebae and plasmodia express different myosin, fragmin
and profilin genes (Kohama & Ebashi, 1986; Kohama e t
a/., 1986; Uyeda & Kohama, 1987; Uyeda e t al., 1988;
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Ph_ysarumpohcephaltlm plasmodium development
Binette e t al., 1990). In addition, the two cell types utilize
different members of the tubulin multigene family (Table
1; reviewed in Burland e t al., 199310); a l - , a3- and B1tubulin isotypes are detectable in amoebae, whereas in
plasmodia a l - , a2-, pl- and P2-tubulin isotypes are
observed.
Genetic control of plasmodium development
Sexual development is the norm in natural isolates of P.
po4cephalnm and leads to the formation of a diploid
plasmodium. Plasmodium formation is under the control
of three unlinked mating-type loci, matA, matB and matC
(reviewed in Dee, 1987). Multiple alleles of all of these
genes exist in natural populations (Collins & Tang, 1977;
Kirouac-Brunet e t al., 1981; Kawano e t al., 1987b). The
mating-type genes matB and matC influence the frequency
with which pairs of amoebae fuse (Dee, 1978; Shinnick et
al., 1978; Youngman e t al., 1979, 1981; Kawano e t al.,
1987b). Amoebae carrying different alleles of matB are
100-1000-fold more likely to fuse than amoebae which are
homoallelic for this locus (Youngman e t al., 1981), while
amoebae which differ at matC can fuse over a greater pH
range than amoebae carrying identical matC alleles
(Shinnick e t al., 1978; Kawano e t al., 1987b).
Amoebae have to acquire the ability to fuse with
genetically compatible amoebae before plasmodium formation can occur. For example, when two strains of
compatible amoebae are mixed and plated out, a period of
amoebal multiplication precedes the onset of fusions,
during which the amoebae become fusion competent
(Pallotta e t al., 1979; Youngman e t al., 1979). Ability to
undergo fusion with a compatible amoeba can also be
acquired during exponential growth in clonal culture
(Shipley & Holt, 1982); if both strains of amoebae are
fusion competent at the time of mixing and plating out,
mating can occur without a time lag. Dense cultures of
amoebae can induce competence in overlying sparse
cultures separated by 0.2 pm filters (Pallotta e t al., 1979;
Shipley & Holt, 1982), suggesting that an extracellular
inducer is responsible for the acquisition of fusion
competence. Preliminary analyses suggest that this inducer may be a complex molecule that acts over a short
range and decays quickly (Nader e t al., 1984). Addition of
partially purified inducer to mixtures of compatible
amoebae advances the start of plasmodium formation
(Nader e t al., 1984).
The third mating-type gene, m a t A (Dee, 1960, 1987), has
no effect on amoebal fusion but controls subsequent
development of the fusion cell (Youngman e t al., 1981).
When two amoebae carrying the same matA allele fuse,
development is blocked before nuclear fusion. If, however, the fusing amoebae carry different alleles of matA,
nuclear fusion occurs in interphase giving rise to a diploid
zygote. After a period of growth, the zygote becomes
binucleate by mitosis unaccompanied by cytokinesis.
Larger diploid plasmodia arise rapidly as a result of
further mitoses unaccompanied by cytokinesis, and
fusions between genetically identical plasmodia (Bailey e t
al., 1990).
Mutations at matA give rise to apogamic strains in which
haploid plasmodia arise within colonies of haploid
amoebae, indicating that the diploid state is not necessary
for plasmodium formation (Cooke & Dee, 1974; Shinnick
etal., 1983). All the apogamic strains currently used show
temperature-sensitive development; plasmodia will develop in colonies of apogamic amoebae cultured at low
temperature (21-22 "C) but not at high temperature
(28-30 "C). At the non-permissive temperature for development, apogamic strains are still able to mate,
showing the same matA-specificity as the strain from
which they were originally isolated (Anderson, 1979).
The gadA (greater asexual development) mutations permitting apogamic development are dominant and genetically inseparable from matA. Since they do not alter
mating-specificity, they are assumed to define a second
function of this complex locus (Anderson, 1979; Anderson e t al., 1989).
As in sexual development, apogamic amoebae undergo a
period of proliferation before clonal plasmodium formation is initiated (Youngman e t al., 1977). The proliferating amoebae secrete a chemical inducer and plasmodium formation is triggered when the inducer reaches
a critical concentration in the local environment (Youngman e t al., 1977). Addition of partially purified inducer to
cultures of apogamic cells advances the initiation of
plasmodium formation (Nader e t al., 1984). The same
preparations also affect mating (see above), suggesting
that the same inducer affects the acquisition of fusion
competence in sexual development and initiation of
apogamic development (Nader e t al., 1984).
Apogamic strains have proved valuable for identifying
developmental mutants ; since the cells remain haploid
throughout development, even recessive mutations can
easily be detected. Mutations affecting development can
be identified by allowing mutagenized apogamic amoebae
to develop at low temperature and screening for colonies
in which plasmodium development is abnormal. The
temperature-sensitivity of apogamic development permits
the use of classical genetic techniques on the mutant cells ;
they can be crossed with compatible strains at high
temperature. The npf (no plasmodium formation) mutations so far identified are recessive and fall into two
groups. The first group, comprising the np$B and npfC
mutations, block the initiation of development and are
genetically inseparable from matA and gadA (Anderson,
1979; Anderson e t al., 1989). Complementation analysis
indicates that these mutations define two further functions
at matA (Anderson e t al., 1989), bringing to four the
number of functions that have been defined at this
complex locus. The second group of npfmutations are
unlinked to matA and show a wide variety of phenotypes
(see below).
Cellular changes during plasmodium
development
The alterations in gene expression, cell organization and
behaviour that accompany plasmodium formation are
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J. B A I L E Y
initiated during a short period in development, but are
not completed for several cell cycles. The majority of
studies of these alterations have been carried out using
apogamically developing cells, although studies on sexual
development indicate that the sequence and timing of
events are very similar in both types of development.
Apogamic development
Time-lapse cinematography of development in the apogamic strain CL (matA.2-derived) shows that a single
haploid amoeba can develop into a plasmodium without
cell or nuclear fusion (Fig. l a ; Bailey e t al., 1987). Either
one or both daughter cells from an apparently normal
amoebal division can develop, indicating that the decision
to divide or develop is not made until after the mitosis at
which a cell is born. The first indication that an amoeba
will develop is when it fails to divide at the end of an
amoebal cell cycle. Instead, the developing uninucleate
cell continues to grow for a period about 2.3-times as long
as a normal amoebal cell cycle. At the end of this extended
cell cycle, the uninucleate cell is twice as large as an
amoeba at mitosis and becomes binucleate by mitosis
without cytokinesis (Anderson e t al., 1976; Bailey e t al.,
1987). About halfway through the extended cell cycle, the
developing cell becomes committed to development and
loses the ability to undergo the amoeba-flagellate transformation (Fig. l a ; Blindt e t al., 1986; Bailey e t al., 1987).
At commitment, the developing cell becomes independent
of the inducer and is able to develop into a plasmodium
when re-plated at low density (Youngman e t al., 1977).
After binucleate cell formation, larger plasmodia arise
rapidly as a result of further nuclear divisions and fusions
between plasmodia. Apogamic plasmodia are able to form
spores of normal appearance but viability is very low
since, with the exception of the occasional diploid nucleus,
the nuclei in the plasmodium are haploid. Similar analyses
of apogamic development in the matA3-derived strain
RA376, show the same sequence of events (Bailey e t al.,
1992).
Nuclear volume increases during the long cell cycle in
apogamic development, but returns to the haploid level
by the time the developing plasmodium is quadrinucleate.
This alteration in nuclear volume is not due to a transient
increase in DNA content (Bailey e t al., 1987), but may be
related to an increase in the RNA content of the
developing uninucleate cell (Larue e t al., 1982), caused by
a burst of RNA synthesis from developmentally regulated
genes. In vegetative cells of P. pabcepbalzlm, there is no G1
phase following mitosis; the 2-3 h S phase follows
immediately after mitosis and the remainder of the cell
cycle is occupied by G2 phase (reviewed in Burland e t al.,
1993b). Measurement of DNA content in developing
uninucleate cells gives no indication of a G1 phase,
indicating that the elongation of the cell cycle is due to an
extension of G2 (Bailey e t al., 1987).
Cell cycle regulation in P. pobcepbalzlnz apparently operates
by a size-control mechanism with mitosis being triggered
when the protein:DNA ratio reaches a particular level
2358
(Laffler & Tyson, 1986). During the long cell cycle, the
developing uninucleate cell grows for an extended period
without any increase in DNA content, suggesting that the
protein:DNA ratio is greater at mitosis in large uninucleate cells than in amoebae (Bailey e t al., 1987). The
presence of a short cell cycle in the binucleate cell (0.7 x an
amoebal cell cycle, Fig. l a ; Bailey e t al., 1987) during
which cell volume and, presumably protein content, do
not double, may serve to return the protein :DNA ratio to
the usual level. Thus, the normal size-control mechanism
which regulates mitosis is altered in developing uninucleate cells in a way that is unknown at present.
Many of the changes in cellular organization, behaviour
and gene expression that accompany development are
initiated during the extended cell cycle. For example,
during the second half of the long cell cycle, the
developing uninucleate cell acquires plasmodial characteristics such as the ability to ingest amoebae and to fuse with
genetically identical plasmodia (Fig. la). It is at this stage
that the plasmodial myosin heavy-chain protein is first
present, as are the 18 kDa plasmodial myosin light-chain
protein (Uyeda & Kohama, 1987) and plasmodial fragmin
proteins (Uyeda e t al., 1988). The amoeba-specific a3tubulin isotype starts to disappear from apogamic cells
during the extended cell cycle, and the plasmodiumspecific @-tubulin isotype is first detected in uninucleate
developing cells (Solnica-Krezel e t al., 1988, 1990). In
addition, a high proportion of cell-type-specific genes
alter their pattern of expression during the long cell cycle
(Sweeney e t al., 1987).
Microtubule organization during apogamic
development
As discussed above, amoebae and plasmodia differ in the
type of mitotic spindle formed. The spindle poles of the
astral amoebal mitoses contain a3-tubulin, whereas plasmodial spindles do not. Conversely, the @-tubulin isotype
is present in the spindle microtubules of plasmodial
mitoses but is absent in amoebae (Solnica-Krezel e t al.,
1991). In most developing uninucleate cells, the mitosis at
the end of the long cell cycle is of the intranuclear
plasmodial type. Other types of spindle are found in some
developing cells at this mitosis, however, indicating that
there may be more than one possible route for the
transition from uninucleate to binucleate cell (SolnicaKrezel e t al., 1991). The mitosis which leads to the
formation of a quadrinucleate cell from a binucleate one is
always of the plasmodial type, indicating that the transition from amoebal to plasmodial mitosis is complete by
this time (Solnica-Krezel e t al., 1991).
In many uninucleate developing cells undergoing plasmodial mitosis, an extranuclear, a3-tubulin-positive
MTOC is present; this appears to be a remnant of the
amoebal one, indicating that the plasmodial intranuclear
MTOC is a different structure (Solnica-Krezel e t al., 1991).
Binucleate cells contain from zero to two MTOCs,
indicating that the amoebal MTOC sometimes duplicates
at mitosis in developing uninucleate cells even though it
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Plysartlm po&-ephaltlm plasmodium development
(a)
Amoebal cell cycle
Open
mitosis
Activation of plasmodium-specific and transiently expressed genes
w
Acquisition of plasmodial characteristics,
e.g. ingestion of amoebae, plasmodial fusions.
npfA and npfG
ene products
irst required
npfE npfK, npfL and
npfM gene products
first required
9.
-
Closed
mitosis
Closed
mitosis
I
Commitment
I
Extended cell cycle
+
2.3 units
0.7units
Amoebal cell cycle
t
+ +
Fusion Diploid
cell
zygote
Closed
mitosis
t
Closed
mitosis
t
Commitment
-
Acquisition of plasmodial characteristics,
e.g. ingestion of amoebae, plasmodial fusions.
m
Extended cell cycle
2.3 units
Fig. 1. Sequence of events in relation t o the cell cycle in (a) apogamic and (b) sexual development.
generally does not form the spindle poles (Solnica-Krezel
etal., 1990). Thus, the amoeba1 MTOC appears to lose the
ability to form the basal bodies of the flagella and the poles
of the spindle, before losing the ability to duplicate at
mitosis and nucleate microtubules. In addition, there is no
strict correlation between the change in expression of the
a3- and @-tubulin isotypes and reorganization of the
cytoplasmic microtubules, suggesting that changes in
isotype usage are not sufficient to bring about alterations
in microtubule organization (Solnica-Krezel e t al., 1990).
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J. B A I L E Y
Sexual development
Analyses of the cellular events of sexual development
reveal that, after zygote formation, the sequence of events
is very similar to those seen in apogamic development
(Fig. 1b). Fusion can occur between any two cells carrying
different alleles of matB and matC, and both amoebae and
flagellates are able to undergo fusion (Ross, 1957; Bailey
et a/., 1990). Fusion occurs at any stage of the cell cycle,
except during mitosis and for a period of about 20 min
thereafter (Bailey e t al., 1990). In matA-heteroallelic
fusion cells, commitment coincides with cell or nuclear
fusion (Fig. l b ; Shipley & Holt, 1982), and ability to
undergo the amoeba-flagellate transformation is lost
shortly after this event (Bailey e t al., 1990). Nuclear
fusion, the first observable event of development, occurs
in interphase about 2 h after cell fusion and appears to
result from microtubule-mediated processes (Bailey e t al.,
1990). Since amoebal fusion does not occur at any
particular stage of the cell cycle, the nuclei in the fusing
cells may be at different stages of the cell cycle. The 2 h
gap between cell- and nuclear fusion would be sufficient
to allow a nucleus that was in S phase at the time of fusion
to complete S and enter G2, ensuring that, by the time of
nuclear fusion, both nuclei were in G2.
As in apogamic development, the developing uninucleate
cell, in this case the diploid zygote, becomes binucleate by
mitosis unaccompanied by cytokinesis at the end of an
extended cell cycle (Fig. l b ; Bailey e t al., 1987, 1990).
There is no evidence for the presence of a G1 phase in the
cell cycle of zygotes ;the extension of the cell cycle appears
to result from the lengthening of G2. At binucleate
plasmodium formation, the zygote is about four-times the
size of an amoeba at mitosis, with an enlarged nucleus but
only diploid DNA content (Bailey e t al., 1990): thus the
protein :DNA ratio alters during sexual development in
the same way as it does in apogamic development. During
the long cell cycle, the developing zygote acquires
plasmodial characteristics such as the ability to ingest
amoebae and to undergo plasmodial fusions (Fig. l b ;
Bailey e t a/., 1990).
Fusion cells, containing two haploid nuclei, generally
have one MTOC associated with each nucleus, one from
each of the fusing cells (Bailey e t al., 1990). By the time
nuclear fusion has occurred, there is a single MTOC
associated with the diploid zygote nucleus. In some
zygotes, two pairs of centrioles appear to be present at the
MTOC, suggesting that the zygote MTOC results from
the fusion of the two amoebal MTOCs (Bailey e t al.,
1990). As in apogamic development, the mitosis at the end
of the extended cell cycle is usually of the intranuclear
plasmodial type and the amoebal MTOC, which is often
present in the cytoplasm during this mitosis, is still
capable of duplicating and nucleating a microtubule
network in the next interphase (Bailey e t al., 1990). Thus,
the switch from amoebal to plasmodial microtubule
organization, as well as all the other cellular events
studied, occur in a very similar way in both sexual and
apogamic development. These observations indicate that
the g a d A mutation causing apogamic development by-
2360
passes the normal requirement for cell- and nuclear fusion
and allows development to occur in a single haploid cell,
without significantly altering the timing of the cellular
events of plasmodium formation.
In fusion cells which are homoallelic for matA, nuclear
fusion does not occur in interphase and the microtubule
cytoskeletons contributed by the fusing amoebae remain
separate (Bailey e t al., 1990). In most cases, the fusing
amoebae pull apart shortly after fusion, but in about 25 %
of cases, the fusing amoebae remain as a single binucleate
fusion cell until amoebal mitosis occurs without any
lengthening of the cell cycle (Bailey etal., 1990). Mitosis in
matA-homoallelic binucleate fusion cells often results in
spindle fusion and the formation of diploid amoebae
(Bailey e t al., 1990). Thus, the presence of two nuclei in a
single cell is not sufficient to trigger interphase nuclear
fusion, or the subsequent events of development, suggesting that these events are under the control of matA.
lnheritance of mitochondria
The mitochondrial DNAs (mtDNAs) from different
isolates of P. po&ephalum contain restriction enzyme site
polymorphisms, allowing them to be identified with ease
(Kawano e t al., 1987a). Plasmodia formed by crossing
amoebae of different mtDNA types normally possess only
one of the two mtDNA types, indicating that inheritance
is uniparental (Kawano e t al., 1987a). Inheritance of
mitochondria is independent of matB and matC, but is
under the control of m a t A or a closely linked locus
(Kawano & Kuroiwa, 1989). The alleles of matA appear
to form a hierarchy such that the mtDNA present in a
plasmodium is that from the amoebal strain carrying the
matA allele of higher status (Kawano & Kuroiwa, 1989;
Meland e t al., 1991). When amoebae of different matA and
mtDNA type are crossed, elimination of one parental
mtDNA type is virtually complete by the time the
developing plasmodia are quadrinucleate, suggesting that
the lower status mtDNA is actively eliminated (Meland e t
al., 1991).
Some amoebal strains carry a linear 16 kb mitochondrial
plasmid (mzj). In crosses where one strain carries this
plasmid, the mtDNA is not uniparentally inherited
(Kawano e t al., 1991). Instead, several mitochondria fuse
into a large mitochondrium containing multiple
mtDNAs ; fusion is followed by several mitochondrial
and mtDNA divisions. Recombination occurs during
these divisions, resulting in mtDNA with a novel
restriction enzyme digestion pattern ; this mtDNA is
passed to the normal mitochondria as they reform. The
mif plasmid is transmitted unaltered to all mitochondria
and subsequently to all amoebal progeny of the plasmodium (Kawano e t al., 1991).
Plasmodium development in strains carrying
maw-unlinkednpf mutations
Strains carrying matA-unlinked npfmutations are blocked
in development after initiation and show a wide variety of
phenotypes (e.g. Wheals, 1973; Anderson & Dee, 1977;
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PLyarzm pohcephaltlm plasmodium development
Bailey e t al. , 1992; Solnica-Krezel e t al. , 1995). Amoeba1
growth is apparently normal in strains carrying these
mutations, suggesting that the npf genes are activated
during development and, thus, are directly or indirectly
under the control of matA. It is possible that some of the
npf genes are required for only a short time during
development, while others are required throughout
plasmodial growth; for most of the mutants, however,
the evidence does not allow one to distinguish between
these possibilities. The matA-unlinked npf mutations
result in blocks at various times during development.
Mutations causing early blocks in plasmodium
development
In cultures of apogamic cells carrying mutations in either
npfA or npjG, the majority of cells do not initiate
development, indicating that these mutations block apogamic development very early and suggesting that the
wild-type product of both these genes is required at, or
very close to, the time of initiation of development (Fig.
l a ; Solnica-Krezel et al., 1995). In developing cultures of
both strains, a few cells show positive markers of
development (such as becoming binucleate, or staining
for fl-tubulin) but plasmodia rarely form, indicating that
cells that initiate development eventually die. In cultures
of cells carrying a mutation at npfA, non-revertant
plasmodia form at low frequency indicating that the
mutation is ‘leaky’ (Anderson & Dee, 1977; SolnicaKrezel e t al., 1995). Although the npfA and npfG
mutations block apogamic development, neither affects
sexual development even in a homozygote (SolnicaKrezel e t al., 1995); the reason for this is unknown at
present.
Mutations causing later blocks in plasmodium
development
The second group of matA-unlinked npf mutations affect
the later stages of plasmodium formation and block both
sexual and apogamic development (np$F, npfK, n p f z ,
npfM; Bailey e t al., 1992; Solnica-Krezel e t al., 1995).
During development in mutant strains, abnormalities are
observed at the end of the long cell cycle, about the time
of binucleate cell formation, suggesting that the wild-type
genes are first required at or before this time (Fig. l a ;
Bailey e t al., 1992; Solnica-Krezel e t al., 1995). This is
consistent with previous studies implicating the second
half of this cell cycle as a crucial one for development,
when many alterations in gene expression and cellular
organization begin (e.g. Sweeney e t al., 1987;Bailey e t a/.,
1987; Solnica-Krezel e t al., 1990). The terminal phenotypes in cells carrying these npf mutations are very
different (Bailey e t al., 1992; Solnica-Krezel e t al., 1995).
In most of these npfmutants, some aspects of development
continue normally while others are blocked, indicating
that the events are not dependent on one another,
although they occur at the same time. For example, cells
carrying a mutation in the npJx gene show normal
microtubule re-organization, even though the abnormal
structure of the mutant cells is gradually developing
(Solnica-Krezel e t al., 1995). Cells carrying a mutation in
n p f z are still able to undergo plasmodial fusions even
though the nuclei have become defective (Bailey e t al.,
1992). Events that are not dependent on one another may
be on different developmental pathways ;evidence for the
existence of such pathways comes from studies of double
mutants. For example, the n p f l n p f z double mutant has
characteristics of both single mutants, indicating that
these two mutations act in different developmental
pathways. The npJF npfG double mutant, however,
resembles npfG in phenotype, suggesting that, in this case,
the wild-type genes act sequentially in the same pathway
(Solnica-Krezel e t al., 1995). These observations suggest
that the various developmental pathways function semiindependently and some pathways may continue for some
time even though other pathways are blocked.
In four of the npfmutants examined so far (np$F, npfG,
n p f i , npfM ), the developing cells eventually die in the
same characteristic manner ; the nuclei condense, lose
their nucleoli and stain intensely with the DNA dye 4’,6diamidino-2-phenylindole (DAPI) (Bailey e t al., 1992;
Solnica-Krezel et a/., 1995). These features are characteristic of death by apoptosis (Kerr e t al., 1972), a process
of cell death observed during morphogenesis in many
eukaryotic systems (e.g. Hengartner & Horvitz, 1994). In
P. po&epbalt/m, death may result from an inability to
complete development, regardless of the primary lesion.
Since apoptosis is often considered to have evolved in
multicellular organisms (Vaux e t al., 1994), these observations of apoptosis-like cell death in a unicellular organism
are interesting. Investigations into the role of cell death in
P. po&epbalum are continuing and may help to elucidate
whether this system is of value to the developing
plasmodium.
The mating-type loci
Any models for the action of the mating-type loci in P.
po&epbalHm must account for the following observations :
(i) heteroallelism at matB and matC increases the frequency
with which pairs of amoebae fuse; (ii) both acquisition of
fusion competence in sexual development and initiation
of apogamic development require the inducer; (iii) the
presence of two different matA alleles in a cell triggers
development ; (iv) the gadA, npfB and npfC mutations all
map at the matA locus and affect the initiation of
development.
The mating-type loci in natural populations
In many fungi, successful development requires heteroallelism at both of two mating-type loci (e.g. Banuett,
1992) but in P. po&epbalt/m, heteroallelism at three
multiallelic loci optimizes the efficiency of development.
If development occurred only between amoebae heteroallelic at matA, matB and matC, an amoeba would be
compatible with 12.5 % (1 in 8) of the progeny from the
same plasmodium and in-breeding between sibling
amoebae would be infrequent. The level of compatibility
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2361
J. B A I L E Y
could reach 50 %, however, as only matA has an absolute
effect on development and amoebae that are heteroallelic
for m a t A and homoallelic for matB and matC give rise to
plasmodia at low frequency. The actual level of compatibility is probably between these two estimates since all
plasmodia so far isolated have carried two alleles of matA
and matB, suggesting that, in the wild, heteroallelism at
these loci is important for plasmodium formation
(Kirouac-Brunet e t a/., 1981); not enough isolates have
been tested for the same conclusion to be drawn about
matC.
The presence of multiple alleles at all three mating-type
loci promotes out-breeding between unrelated amoebae.
So far, 14 matA, 13 matB and 3 matC alleles have been
identified (Collins & Tang, 1977; Kirouac-Brunet e t a/.,
1981; Kawano e t a/., 1987b), but many more alleles may
exist since every isolate so far tested has carried two
additional alleles of matA and matB (Kirouac-Brunet e t
al., 1981); few isolates have been tested for matC. It is not
known, however, whether each natural population contains a unique set of alleles or whether the same allele
occurs in geographically distant populations. We also
have no information about how the flow of individuals
between populations might influence the number of alleles
of each gene present in a population.
Mode of action of matB and matC
The MatC protein strongly influences the frequency of
amoebal fusion under specific pH conditions, suggesting
that the matC product is attached to the outer membrane
of the amoebae and contains ionizable groups on its
surface. The matB product influences amoebal fusion over
a larger range of conditions than MatC and is probably
also located on the amoebal surface. The MatB protein
may function as a receptor, linking events at the cell
surface to gene action in the nucleus and could be
involved in one or more of the following processes:
recognition of matB product on adjacent amoebae;
holding amoebae together prior to fusion ; or subsequent
membrane fusion. The mechanisms by which heteroallelism at matB and matC enhance amoebal fusion are
unknown. These genes could be constitutively expressed,
but may be switched on in response to external stimuli. If
matB is constitutively expressed, the inducer could bring
about fusion competence by altering the conformation of
the MatB protein to an active form, capable of interacting
with the matB products of other amoebae. If matB and
matC are not constitutively expressed, the inducer may
activate these genes, leading to the appearance of their
products on the amoebal surface and the acquisition of
fusion competence.
Structure and mode of action of matA
The m a t A locus initiates plasmodium development and
controls the accompanying alterations in gene expression
and cellular organization ; it is presumably a transcription
factor, or a crucial part of a transcription factor complex.
This locus could be directly responsible for all developmental alterations in gene expression but, since plas-
2362
modium development involves a number of parallel
pathways, matA probably initiates a cascade of gene
action involving other transcription factors and structural
genes. In this case, the promoters of amoeba-specific
genes, and genes at the start of the developmental
pathways, should contain conserved motif(s) directing
binding of the MatA protein. Two candidates for genes
directly activated by m a t A are n p f A and npfG ; both block
development very close to initiation and, thus, could be at
the start of developmental pathways. It is not known if
matA is expressed constitutively, or if it is switched on in
response to an external stimulus. m a t A might be switched
on by the inducer when matB and matC are activated, but
could also be activated later as a result of the acquisition
of fusion competence. If the inducer activates all three
mating-type genes simultaneously, then it could also
switch on the mutant m a t A allele in apogamic strains,
thus permitting development in a single haploid cell.
Although several mutations at matA have been identified
by classical genetic analysis, the molecular basis of the
mode of action of matA is unknown. It has been suggested
that mating-type interactions in multiallelic systems
depend upon specific tight interactions between the
products of a single allele (Anderson & Holt, 1981).
Recent studies in basidiomycete fungi have revealed how
several such systems operate at a molecular level. In the
smut fungus Ustilago mqdis, for example, the a locus
mediates fusion of the haploid cells while the multiallelic
b locus governs subsequent development into the pathogenic filamentous dikaryon (Banuett, 1992). The b locus
contains two divergently transcribed genes, bE and b W ,
which are not homologous to each other although there is
homology between the bE and bW genes from different b
alleles. Both genes have a variable N-terminal domain and
a highly conserved C-terminal constant domain containing a DNA-binding homeodomain motif (Banuett,
1992). The bE and bW proteins from one b locus are
presumed to form a heterodimer in which the DNA
recognition site is inaccessible, and development is not
initiated. The heterodimers formed between the bE and
bW proteins from different b alleles can bind DNA
leading to the activation of development-specific genes
and initiation of development (Banuett, 1992).
The U. mqdis system serves as a useful model for the
mode of action of matA in P. po&phahm since specific
mutations in a pair of genes like bE and bW could lead to
thegadA, np$B and npfCclasses of mutations. For example,
apogamic strains would result from a gadA mutation in
one of the genes, allowing the formation of an active
heterodimer in a haploid cell at the permissive temperature. Mutations in either np$B or npfC would knock
out one of the paired genes, preventing heterodimers
from forming and blocking development (R. W.
Anderson, personal communication). Although this
model can explain the genetic evidence for the control of
development in P. pobcephalmz, since the basidiomycetes
are evolutionarily distant from the myxomycetes, the
molecular basis of the P. po&ephalm system may be
totally different. Until matA has been cloned and sequenced it will not be possible to determine how it operates.
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Pbysartlm po&ephaltlm plasmodium development
DNA transformation
Reporter gene constructs based on the bacterial chloramphenicol acetyltransferase and firefly luciferase genes
(Burland e t al., 1992; Bailey e t al., 1994) have been used to
optimize transformation protocols, to study promoter
function, and to ascertain the effect of D N A fragments on
the stability of introduced D N A and the efficiency of
stable transformation. Stable transformation vectors containing the hygromycin phosphotransferase gene under
the control of a P. po&$dmz
actin promoter as a
selectable marker have been developed (Burland e t a/.,
1993a). Similar vectors were recently used to carry out the
first gene disruption in P. po&ephaltlm (Burland &
Pallotta, 1995); further gene disruptions are sure to
follow. As transformation levels improve, it will become
possible to clone genes by complementation ; candidate
genes for such an approach include matA and the npf
mutants.
Anderson, R. W., Hutchins, G., Gray, A., Price, J. & Anderson, 5.
E. (1989). Regulation of development by the matA complex locus in
Pbysarum po&epbalum. J Gen Microbioll35, 1347-1 359.
Bailey, J., Anderson, R. W. & Dee, J. (1987). Growth and
development in relation to the cell cycle in Pbysarum pobcepbalum.
Protoplasma 141, 101-1 11.
Bailey, J., Anderson, R. W. & Dee, J. (1990). Cellular events during
sexual development from amoeba to plasmodium in the slime
mould Pbysarum pobcepbalum. J Gen Microbioll36, 739-751.
Bailey, J., Solnica-Krezel, L., Anderson, R. W. & Dee, J. (1992). A
developmental mutation (np$ I) resulting in cell death in Pbysarum
pobcepbalum. J Gen Microbiol 138, 2575-2588.
Bailey, J., Benard, M. & Burland, T. (1994). A luciferase expression
system for Pbysarum that facilitates analysis of regulatory elements.
Curr Genet 26, 126-1 31.
Banuett, F. (1992). U.ttiIago maydis, the delightful blight. Trends
Genet 8, 174-179.
Binette, F., Benard, M., Laroche, A., Pierron, G., Lemieux, G. &
Pallotta, D. (1990). Cell-specific expression of a profilin gene family.
D N A Cell Biol9, 323-334.
The future
The development of a D N A transformation system for P.
po&cephalzm was a crucial step in completing the range of
molecular techniques required for the study of development in this organism. A combination of molecular
genetic techniques, classical genetics and cellular analyses
will allow us to answer many key questions about the
control of development in P. poEycephalzjm. This multifaceted approach should lead to the elucidation of the
mechanisms by which development is initiated and how
changes in gene expression and cellular organization are
controlled during the amoebal-plasmodia1 transition.
Acknowledgements
I would like to thank Drs Jennifer Dee and Roger Anderson for
sparking my interest in Pbysarum development, for their helpful
comments and useful insights on this manuscript, and for their
past and continuing support of my career. I would also like to
thank the following for financial support: SERC (grant no.
GR/D34530) ; The University of Wisconsin Graduate School ;
Programme Project grant CA23076 and core grant CA07175
from the National Cancer Institute; The Wellcome Trust (grants
034879 and 042524). Finally, I would like t o thank my colleagues
in the Pbysarum field who have been generous with time,
discussions and sharing unpublished findings.
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