DEVELOPMENTAL
BIOLOGY
47, 97-105 (19%)
The Amoeba1 Cell of Physarum polycephalum: Formation and Growth DAVID McArdle
N. JACOBSON' AND WILLIAM
Laboratory,
University
of Wisconsin,
Madison,
Colony
F. DOVE Wisconsin 53706
Accepted June 3, 1975 Two stages of colony growth were observed during microscopic studies of Physarum polycephalum amoebae. During the first stage, “spreading growth,” the colony is composed of dispersed single cells. During the second stage, “aggregate growth,” most of the active cells in a colony are aggregated in a ring at the colony boundary. Measurements of cell movement as a function of bacterial concentration indicate that, during both spreading and aggregate growth, cell movements are not affected by changes in bacterial concentration but that the transition from spreading to aggregate growth occurs earlier on plates with lower bacterial concentrations. These results indicate that autonomous characteristics of the amoebae are more important for the determination of colony form than local variations in the concentrations of nutrients. The genetic determination of colony form is demonstrated by the existence of mutants that display specific alterations in colony morphology. Because the aggregate rings of these mutants move at an increased rate, mutant clones appear as variant sectors of wild-type colonies. The increased rate of mutant ring movement suggests that this selection method may be a useful technique for isolating mutant myxamoebae with defects in movement and behavior. INTRODUCTION
The myxomycete Physarum polyceph,alum is an organism that offers opportunities for studies of the molecular mechanisms involved in eukaryotic cell movement. During the different stages of its life cycle, P. polycephalum exhibits a variety of motile phenomena including rapid protoplasmic streaming, amoeboid movement, flagellar propelled swimming, and the protoplasmic movements involved in the formation of stalked sporangia (Gray and Alexopoulos, 1968). Moreover, plasmodia are known to contain large quantities of actin and myosin, and techniques are available to purify these proteins in milligram quantities from plasmodial homogenates (Adelman and Taylor (1969). Finally, the results of recent studies of the genetic system of P. polycephalum suggest that the techniques of molecular genetics can be combined with phenomenological and biochemical techniques to study the molecular basis of I Present address: Department of Anatomy, Duke University Medical School, Durham, N. C. 27710.
the motile phenomena of P. polycephalum (Dee, 1966; Haugli and Dove, 1972). Processes that involve movement in response to a stimulus are often particularly suitable for genetic studies because of the possibility of direct selection of mutants. These selections are generally achieved by isolating deviant individuals that become spatially separated from the wild type because they fail to respond properly to a stimulus. Selection procedures of this kind have been used successfully to isolate mutants of Escherichia coli (Armstrong et al., 19671, Drosophila melanogaster (Benzer, 1967) and Paramecium aurelia (Kung, 1971). This communication presents the results of some initial studies directed toward the development of procedures of this kind for the selection of mutant P. polycephalum amoebae with defects in cell movement and behavior. MATERIALS
Culture
AND
METHODS
Conditions
(a) Mynamoebae. Amoebae were tured on lawns of live or formalin-killed
culE.
98
DEVELOPMENTAL BIOLOGY
VOLUME 47, 1975
coli on agar or on Millipore filters (Haugli was constructed on the principle of first and Dove, 1972). Three different media selecting for segregants of Wis 1 which were used for the agar plates: 0.05% Oxoid mated, sporulated, and germinated effiliver infusion (LIA); the semidefined me- ciently and then carrying two such segredium of Daniel and Baldwin (19641, diluted gants through a series of offspringtenfold (SD/lo); and McIlvaine’s buffer at younger parent inbreedings. pH 5, diluted tenfold (pH 5) (Collins and The first step was accomplished by platTang, 1973). ing amoebae issuing from Wis 1 on SD/l0 (b) Matings. Matings were carried out plates at concentrations from 10” to 10” per by inoculating with toothpicks amoebae of plate. Forty microplasmodia were picked different mating type onto a pH 5 agar as soon as they were visible, and each was plate spread with 10’” formalin-killed bac- tested for ability to sporulate and germiteria. nate. The best plasmodium was then used (c) Plasmodia. Microplasmodia were to initiate a second cycle of selection just transferred from the mating plate to an like the first. From this, two founding parSD/l0 agar plate spread with 10”’ for- ents were picked, DJ128 and DJ129. These malin-killed bacteria. After plasmodia had parents mated well and gave plasmodia begun to grow vigorously they were transthat sporulated and germinated well. Inbreeding was then performed by typferred onto a piece of filter paper (Schleicher and Schuell 576) suspended on ing for mating specificity the amoebae deriving from a plasmodium and then mata stainless-steel grid over diluted semideing an amoebae with its younger parent. fined medium (SD/Z). (d) Sporulation. Plasmodia were al- In this way, the inbred partners DJ437 and DJ620 were produced: lowed to starve on SDI2. One to two days after the initiation of starvation a series of -illuminations was begun. The plasmodia were illuminated 4 hr each day until sporu
BIOLOGY
47, 97-105 (19%)
The Amoeba1 Cell of Physarum polycephalum: Formation and Growth DAVID McArdle
N. JACOBSON' AND WILLIAM
Laboratory,
University
of Wisconsin,
Madison,
Colony
F. DOVE Wisconsin 53706
Accepted June 3, 1975 Two stages of colony growth were observed during microscopic studies of Physarum polycephalum amoebae. During the first stage, “spreading growth,” the colony is composed of dispersed single cells. During the second stage, “aggregate growth,” most of the active cells in a colony are aggregated in a ring at the colony boundary. Measurements of cell movement as a function of bacterial concentration indicate that, during both spreading and aggregate growth, cell movements are not affected by changes in bacterial concentration but that the transition from spreading to aggregate growth occurs earlier on plates with lower bacterial concentrations. These results indicate that autonomous characteristics of the amoebae are more important for the determination of colony form than local variations in the concentrations of nutrients. The genetic determination of colony form is demonstrated by the existence of mutants that display specific alterations in colony morphology. Because the aggregate rings of these mutants move at an increased rate, mutant clones appear as variant sectors of wild-type colonies. The increased rate of mutant ring movement suggests that this selection method may be a useful technique for isolating mutant myxamoebae with defects in movement and behavior. INTRODUCTION
The myxomycete Physarum polyceph,alum is an organism that offers opportunities for studies of the molecular mechanisms involved in eukaryotic cell movement. During the different stages of its life cycle, P. polycephalum exhibits a variety of motile phenomena including rapid protoplasmic streaming, amoeboid movement, flagellar propelled swimming, and the protoplasmic movements involved in the formation of stalked sporangia (Gray and Alexopoulos, 1968). Moreover, plasmodia are known to contain large quantities of actin and myosin, and techniques are available to purify these proteins in milligram quantities from plasmodial homogenates (Adelman and Taylor (1969). Finally, the results of recent studies of the genetic system of P. polycephalum suggest that the techniques of molecular genetics can be combined with phenomenological and biochemical techniques to study the molecular basis of I Present address: Department of Anatomy, Duke University Medical School, Durham, N. C. 27710.
the motile phenomena of P. polycephalum (Dee, 1966; Haugli and Dove, 1972). Processes that involve movement in response to a stimulus are often particularly suitable for genetic studies because of the possibility of direct selection of mutants. These selections are generally achieved by isolating deviant individuals that become spatially separated from the wild type because they fail to respond properly to a stimulus. Selection procedures of this kind have been used successfully to isolate mutants of Escherichia coli (Armstrong et al., 19671, Drosophila melanogaster (Benzer, 1967) and Paramecium aurelia (Kung, 1971). This communication presents the results of some initial studies directed toward the development of procedures of this kind for the selection of mutant P. polycephalum amoebae with defects in cell movement and behavior. MATERIALS
Culture
AND
METHODS
Conditions
(a) Mynamoebae. Amoebae were tured on lawns of live or formalin-killed
culE.
98
DEVELOPMENTAL BIOLOGY
VOLUME 47, 1975
coli on agar or on Millipore filters (Haugli was constructed on the principle of first and Dove, 1972). Three different media selecting for segregants of Wis 1 which were used for the agar plates: 0.05% Oxoid mated, sporulated, and germinated effiliver infusion (LIA); the semidefined me- ciently and then carrying two such segredium of Daniel and Baldwin (19641, diluted gants through a series of offspringtenfold (SD/lo); and McIlvaine’s buffer at younger parent inbreedings. pH 5, diluted tenfold (pH 5) (Collins and The first step was accomplished by platTang, 1973). ing amoebae issuing from Wis 1 on SD/l0 (b) Matings. Matings were carried out plates at concentrations from 10” to 10” per by inoculating with toothpicks amoebae of plate. Forty microplasmodia were picked different mating type onto a pH 5 agar as soon as they were visible, and each was plate spread with 10’” formalin-killed bac- tested for ability to sporulate and germiteria. nate. The best plasmodium was then used (c) Plasmodia. Microplasmodia were to initiate a second cycle of selection just transferred from the mating plate to an like the first. From this, two founding parSD/l0 agar plate spread with 10”’ for- ents were picked, DJ128 and DJ129. These malin-killed bacteria. After plasmodia had parents mated well and gave plasmodia begun to grow vigorously they were transthat sporulated and germinated well. Inbreeding was then performed by typferred onto a piece of filter paper (Schleicher and Schuell 576) suspended on ing for mating specificity the amoebae deriving from a plasmodium and then mata stainless-steel grid over diluted semideing an amoebae with its younger parent. fined medium (SD/Z). (d) Sporulation. Plasmodia were al- In this way, the inbred partners DJ437 and DJ620 were produced: lowed to starve on SDI2. One to two days after the initiation of starvation a series of -illuminations was begun. The plasmodia were illuminated 4 hr each day until sporu
