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Printed in Great Britain 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 2355 . ' 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; Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 2357 . -5 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 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). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 2359 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; Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 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$B or npfC would knock out one of the paired genes, preventing heterodimers from forming and blocking development (R. W. Anderson, personal communication). Although this model can explain the genetic evidence for the control of development in P. pobcephalmz, since the basidiomycetes are evolutionarily distant from the myxomycetes, the molecular basis of the P. po&ephalm system may be totally different. Until matA has been cloned and sequenced it will not be possible to determine how it operates. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 05:47:53 Pbysartlm po&ephaltlm plasmodium development DNA transformation Reporter gene constructs based on the bacterial chloramphenicol acetyltransferase and firefly luciferase genes (Burland e t al., 1992; Bailey e t al., 1994) have been used to optimize transformation protocols, to study promoter function, and to ascertain the effect of D N A fragments on the stability of introduced D N A and the efficiency of stable transformation. Stable transformation vectors containing the hygromycin phosphotransferase gene under the control of a P. po&$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). 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