Flow cytofluorometric analysis of the nuclear division cycle of Physarum polycephalum plasmodia

Abstract

The nuclear cycle kinetics of Physarum polycephalum plasmodia were examined using flow cytofluorometry. The dyes Hoechst 33342 and propidium iodide were used to stain the DNA of isolated nuclei. In asynchronously growing microplasmodia, S phase consists of 13-15% of the nuclear division cycle time. Nuclei isolated from individual macroplasmodia, which have previously been demonstrated to divide in synchrony, were shown to be less synchronized during late S phase than during mitosis. The results obtained demonstrate the feasibility of flow cytometric measurement of the properties of nuclei isolated from a single cell.Key terms: Physarum polycephalum, DNA s*thesis, nuclear division synchrony, Hoechst 33342, flow cytometryThe slime mold Physarum polycephalum has been widely used for studying cell growth and differentiation (15,20). It has been used as a model organism for investigating the cell cycle in eukaryotes mainly because the many nuclei of the large plasmodia (macroplasmodia) obtained in surface cultures exhibit naturally synchronous mitosis (22). Early studies showed that the cell cycle of Physarum is atypical. There is no cytokinesis after nuclear division (22) and the synthesis of L)NA in the plasmodium starts immediately after telophase ( 3 , 18), leaving no G1 phase. Agitation of liquid culture creates plasmodia1 fragments (microplasmodia) . It has been generally agreed that this process eliminates any synchrony between microplasmodia, but that within each microplasmodium the nuclei divided in synchrony (9,20).Because flow cytometry can measure biochemical properties on a cell by cell basis, it has been used extensively for the analysis of cell cycle kinetics of mammalian cells (for historical and technical reviews see reference 14). Methods for measuring the DNA content of unfixed mammalian cells have been developed using the intercalative dye propidium iodide ( 5 , l l ) and the nonintercalative bisbenzimidazole dyes Hoechst 33258 and 33342 (1,12). In order to examine the nuclear division cycle of' Physarum, we have used these dyes for the flow cytometric measurement of the DNA content of purified nuclei from microplasmodia and individual macroplasmodia. Materials and MethodsCulture Methods: P. polycephalum (strain a x i) microplasmodia were grown a t 26°C in shaking flasks essentially as described by Daniel and Baldwin (6). To label DNA of growing nuclei, 'H-methylthymidine (10 pCi/ml, Moravek Biochemicals, City of Industry, CA) was added to the culture medium 2 hr before the isolation of nuclei. Total 'H radioactivity of sorted nuclei was measured by liquid scintillation counting. For preparing synchronously dividing surface macroplasmodia, 0.3 ml of suspended microplasmodia were allowed to coalesce on 7-cm diameter filter papers (6).Preparation and Staining of Nuclei: Nuclei were isolated following previously described procedures (10, 16) with some modifications. Microplasmodia from one shaking flask (50 ml of culture medium) inoculated 56 hr before harvesting were washed twice with 70 ml of ice-c...

Realtime acquisition, storage, and display of correlated three‐parameter flow cytometric data

“…Correlated 2-parameter flow-cytometric data is usually represented with isometric displays, contour maps (17,19) or monochrome scattergrams (4,6,11,18,21,23 about the data's X-Y coordinates, as well. The use of "hidden-colour" suppression in this display reduces the incidence of false colours potentially generated by the overlap of different colour regions.…”

Specific nuclear elimination in polyploid plasmodia of the slime mold physarum polycephalum

“…polyce phalum are unicellular, multinucleate organisms. The plasmodia1 cell cycle is characterized by synchronous nuclear divisions (6,9,15) and the lack of a G1-phase (21). After nutrient deprivation for 3 to 5 days, plasmodia are induced to sporulate by light (4).…”

Flow cytometry reveals a high degree of genomic size variation and mixoploidy in various strains of the acellular slime mold Physarum polycephalum

“…However, chromosomal counts and quantitative Feulgen DNA analysis are technically difficult in this species (14,151. Previous attempts to use flow cytometric analysis lacked internal standard-ization (16,22). In the natural synchronous macroplasmodial phase of its life cycle (4,18,23), Physarurn constitutes a unique model system for the study of cell cycle regulation, DNA replication, and gene expression (19).…”