Physarum Plasmodia Do Contain Cytoplasmic Microtubules!

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Physarum Plasmodia Do Contain Cytoplasmic Microtubules! Physarum plasmodia do contain cytoplasmic microtubules! ISABELLE SALLES-PASSADOR, ANDRE MOISAND, VIVIANE PLANQUES and MICHEL WRIGHT Laboratoire de Pharmacologie et de Toxicologie Fondamentales, C.N.R.S., 205 route de Narbonne, 31077 Toulouse-Cedex, France Summary It has been claimed that the plasmodium of the dense and complex three-dimensional network, dis- myxomycete Physarum polycephalum constitutes a tinct from the microfilamentous domains and from very unusual syncytium, devoid of cytoplasmic the nuclei. The orientation of the microtubule microtubules. In contrast, we have observed a network varies according to the plasmodial domain cytoplasmic microtubule network, by both electron examined. Generally microtubules show no special microscopy and immunofluorescence in standard orientation except in plasmodial veins where they synchronous plasmodia, either in semi-thin sections are oriented parallel to the long axis of the veins. or in smears, and in thin plasmodia, used as a Differences between our observations and those of convenient model. Cytoplasmic microtubules could previous workers who failed to find cytoplasmic be seen after immunofluorescent staining with three microtubules in plasmodia are discussed. We pro- different monospecific monoclonal anti-tubulin anti- pose that they reflect difficulties of observation bodies. The immunolabelling was strictly restricted mainly due to the fluorescent background. In con- to typical microtubules as shown by electron mi- trast with the previous view, the discovery of a croscopy. These cytoplasmic microtubules were en- microtubule cytoplasmic cytoskeleton in Physarum tirely and reversibly disassembled by cold treatment plasmodia raises several questions concerning its and by either of two microtubule poisons: methyl relationships with other cellular organelles and its benzimidazole carbamate and griseofulvin. The dynamics during different cell cycle events. microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight Key words: microtubule, microtubule disassembly, microtubule poisons, griseofulvine, methyl benzimidazole carbamate, microtubules. Cytoplasmic microtubules form a myxomycete, Physarum, syncytium. Introduction electron microscopy (Dugas and Bath, 1962; Rhea, 1966; Porter et al. 1965; Havercroft and Gull, 1983) and The microtubular and microfilamentous cytoskeletonS immunofluorescence staining (Havercroft and Gull, 1983; have been extensively studied in the plasmodium of Solnica-Krezel et al. 1990). In contrast, several lines of Physarum, a large syncytium reaching several cm in evidence have suggested that microtubules might be diameter and containing up to 109 nuclei. This macro- present during interphase. First, although tubulin is scopic cell is able to move on its substratum and exhibits rapidly degraded after mitosis (Carrino and Laffler, 1985; vigorous cytoplasmic streaming (Sachsenmaier et al. 1972) Ducommun and Wright, 1989), the amount of tubulin in cytoplasmic differentiated veins; these movements reaches a plateau at the end of S-phase until the next involve an actomyosin network (Porter et al. 1965; Kessler, cyclic synthesis of tubulin occurring in late G2-phase 1982). Microtubules have been observed only in the (Carrino and Laffler, 1985; Ducommun and Wright, 1989). intranuclear spindle during mitosis (Guttes et al. 1968; Second, the heat-soluble 125 kDa polypeptide, a protein Goodman and Ritter, 1969; Ryser, 1970; Sakai and known to bind to microtubules in vitro, is phosphorylated Shigenaga, 1972; Wille and Steffens, 1979; Havercroft and twice during the cell cycle: at late S/early G2-phase and at Gull, 1983) and, with the exception of one unconvincing late G2/prophase stage (Albertini et al. 1990). The last report (McManus and Roth, 1967), it has been repeatedly phosphorylation event could possibly be correlated with claimed that plasmodia are devoid of cytoplasmic the formation of the mitotic spindle, but the former could microtubules (Green et al. 1987; Burland et al. 1983; Paul not be understood, given the apparent absence of micro- et al. 1987; Burland et al. 1988; Eon-Gerhardt et al. 1981; tubules during interphase. Electron microscopy and Gull and Trinci, 1974; Roobol et al. 1984; Birkett et al. immunofluorescence observations have unambiguously 1985a; Solnica-Krezel et al. 1988; Sauer, 1982; Burland, demonstrated that the intranuclear spindle is nucleated, 1985; Laffler and Tyson, 1985; Anderson et al. 1985; during early prophase, on an intranuclear spindle organiz- Solnica-Krezel et al. 1990). In this respect Physarum ing center (Sakai and Shigenaga, 1972; Blessing, 1972; plasmodia seem to constitute a very unusual eukaryotic Tanaka, 1973), and disappears in less than two minutes at cell. the end of telophase (Goodman and Ritter, 1969; Guttes et Despite these numerous reports, the absence of cytoplas- al. 1968). Thus it is likely that any putative interphase mic microtubules was based upon a few observations by microtubules would be located in the cytoplasm. Journal of Cell Science 100, 509-520 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 509 In this report we demonstrate, using appropriate at —20°C. Then the plasmodial pieces were washed in PBS, conditions for both electron microscopy and immunolabel- embedded in Tissue-Tek (Miles) and frozen in liquid nitrogen. ling, that the cytoplasm of Physarum plasmodia contains a Semi-thin sections (4-6 fun) were obtained, placed on slides covered with 1 % gelatin and processed for immunofluorescence. three-dimensional network of microtubules that has no Third, observation of cytoplasmic microtubules in smears ob- obvious relationship with the nuclei. tained from standard synchronous plasmodia was performed in the following way: plasmodial pieces (2 cm x 2 cm) were cut, with the supporting filter, from standard plasmodia during the third Materials and methods cell cycle, and incubated for 15 min at 17 °C in 10 mM Pipes, pH6.5, 5mM EGTA, 10 mM magnesium chloride, 4 M glycerol, Physarum plasmodia 0.1% Triton X-100. Then, smears were fixed for 10 min in cold Microplasmodia (strains CL, M3CIV, TU291, CH713xCH957, methanol and dried or kept in PBS at 4°C before immunofluor- CH713XLU860) were grown at 22°C in agitated cultures (Daniel escence processing. and Rusch, 1961; Daniel and Baldwin, 1964) and used to prepare synchronous plasmodia (Guttes and Guttes, 1964). Microplas- Immunofluorescence modia were fused for 2h on the center of a nitrocellulose The rat monoclonal antibody YL 1/2 (Kilmartin et al. 1982) was membrane in the absence of nutrients. The resulting standard used for all immunofluorescence observations of microtubules plasmodium, grown on semi-defined agar medium (Wright and unless otherwise stated. Plasmodia were reacted for lh at 37 °C Tollon, 1978), was synchronous as judged by the observation of with anti-tubulin antibodies diluted in PBS containing 25 % fetal mitotic stages (Guttes and Guttes, 1964). Thin plasmodia were calf serum, then washed twice in PBS containing 0.05 % Tween-20 obtained from square plasmodial pieces (3 mm x 3 mm) cut, with and in PBS. Then plasmodia were reacted with goat anti-rat or the supporting membrane, from the growing edge of standard goat anti-mouse antibodies labelled with fluorescein, diluted in plasmodium during the third cell cycle following microplasmodial PBS containing 40 % fetal calf serum, for 30 min at 37 °C and then fusion. Plasmodial pieces were placed on a thin film (1 mm) of 1 % washed as before. Nuclei were stained with DAPI (0.2//g ml"1 in agar spread on a glass slide (Naib-Majani et al. 1983), covered PBS) for 15 min at 37 °C and washed twice for 5 min in PBS. with a cellophane membrane and sandwiched with another sheet Preparations were mounted in Mowiol (Planques et al. 1989) and of 1% agar (thickness 4 mm). After 6h the specimens reached a sealed with paraffin wax. Fluorescein (FITC) and DAPI staining diameter of 1-2 cm and were taken at intervals. A smear was were observed by epifluorescence using a Zeiss Axiophot observed by phase-contrast microscopy (Guttes and Guttes, 1964), microscope equipped with a x40 Plan-neofluar objective (NA, while the remaining part was fixed for immunofluorescence. The 0.90), a x63 Plan-apochromat objective (NA, 1.4) and X100 Plan- number of nuclei in thin plasmodia was determined by immuno- neofluar objective (NA, 1.30), an Optovar varying from xl.25 to fluorescence in a Malassez slide: the plasmodia were homogenized x2.5 and a x4 TV camera adaptor. Images, recorded with a in 0.25 ml 10 mM Tris-HCl (pH7.0), 0.25 M sucrose, 10 mix Nocticon camera (LH 4015 from Lhesa), were digitized with an calcium chloride and 0.1% Triton X-100 with a Dounce image processing system (Sapphire from Quantel). Except when homogenizer. The nuclear suspension was diluted in the same stated otherwise, 200 frames were averaged for each picture, an buffer, incubated in the presence of DAPI (4',6-diamidino-2- out-of-focus image was subtracted from the image to reduce phenylindole: 0.2 fig ml"1 in PBS) for 15min and counted by diffuse fluorescence and the final image was recalculated using epifluorescence. the histogram function. Images stored on an optical laser disk, were photographed on a black and white- monitor with a Nikon 35 mm camera (macro 50 mm lens) using Kodak T-Max film with Plasmodial treatments an exposure time varying from 0.25 to 0.5 s. Alternatively, images Thin sandwiched plasmodia incubated at 0°C were placed on of thin plasmodial strands have been observed with a laser scan crushed ice before subsequent treatment for immunofluorescence. microscope (Zeiss LSM 10) equipped with a x63 Plan-apochromat Treatment with the microtubule poisons methyl benzimidazole objective, a 488 nm argon laser, used at 10 % of its maximal power, carbamate (200/JM in 0.5% dimethyl sulfoxide, final concn) and and a X60 zoom. The fluorescence background was removed from griseofulvine (200 ;<M in 0.5% dimethyl sulfoxide, final concn) each picture, using the histogram function, before the construc- were performed on thin plasmodia grown on cellulose acetate and tion of stereo views.
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