Physarum plasmodia do contain cytoplasmic microtubules!

It has been claimed that the plasmodium of the myxomycete Physarum polycephalum constitutes a very unusual syncytium, devoid of cytoplasmic microtubules. In contrast, we have observed a cytoplasmic microtubule network, by both electron microscopy and immunofluorescence in standard synchronous plasmodia, either in semi-thin sections or in smears, and in thin plasmodia, used as a convenient model. Cytoplasmic microtubules could be seen after immunofluorescent staining with three different monospecific monoclonal anti-tubulin antibodies. The immunolabelling was strictly restricted to typical microtubules as shown by electron microscopy. These cytoplasmic microtubules were entirely and reversibly disassembled by cold treatment and by either of two microtubule poisons: methyl benzimidazole carbamate and griseofulvin. The microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight microtubules. Cytoplasmic microtubules form a dense and complex three-dimensional network, distinct from the microfilamentous domains and from the nuclei. The orientation of the microtubule network varies according to the plasmodial domain examined. Generally microtubules show no special orientation except in plasmodial veins where they are oriented parallel to the long axis of the veins. Differences between our observations and those of previous workers who failed to find cytoplasmic microtubules in plasmodia are discussed. We propose that they reflect difficulties of observation mainly due to the fluorescent background. In contrast with the previous view, the discovery of a microtubule cytoplasmic cytoskeleton in Physarum plasmodia raises several questions concerning its relationships with other cellular organelles and its dynamics during different cell cycle events.

The effects of microinjection of rhodamine-phalloidin on mitosis and cytokinesis in early stage Drosophila embryos.

Rhodamine-phalloidin was microinjected into early stage Drosophila embryos, which were then allowed to develop for various times, fixed, and examined by fluorescence microscopy. A gradient of effects was seen. Close to the site of injection an area of diffuse bright fluorescence was found which included lumps and long strands of fluorescent material. Around this region particular cytoplasmic domains showed a denser F-actin distribution. These domains included the nuclear islands of the preblastoderm, the cortical caps of the syncytial blastoderm, and the contractile ring network which forms during cellularization of the blastoderm. It is proposed that these domains are regions of preferential actin polymerization under the appropriate cellular conditions and that the injected phalloidin causes incorporation of additional polymer into existing structures. Further away the pattern of phalloidin staining corresponded to that found with fixed material. In contrast to the domains of apparent additional F-actin polymerization a reduction of actin incorporated into small aggregates was found, both in syncytial blastoderm stages and during cellularization. This occurred in regions where additional actin had been incorporated into adjacent actin-rich structures. A storage role for the aggregates, which are depleted when F-actin is polymerized, is proposed. Both mitosis and cytokinesis were found to be slowed but the inhibition was only transient. However, most embryos died without differentiating. Rarely, differentiated tissues formed and the musculature was strongly stained by rh-phalloidin. When embryos were injected immediately prior to the start of cellularization cytokinesis was inhibited only locally and continued normally elsewhere. This finding argues against the hypothesis that contraction of an actomyosin network over the whole surface is the only force involved in the cellularization of the blastoderm and that local factors, e.g., plasmalemma extension, must be involved.

Distribution of microtubules containing post-translationally modified alpha-tubulin during Drosophila embryogenesis.

The distribution of microtubules (MTs) enriched in detyrosinated alpha-tubulin (Glu-tubulin) was studied in Drosophila embryos by immunofluorescence microscopy by using a monoclonal antibody (ID5) which was raised against a 14-residue synthetic peptide spanning the carboxyterminal sequence of Glu-tubulin (Wehland and Weber: J. Cell Sci. 88:185-203, 1987). While all MT arrays contained tyrosinated alpha-tubulin (Tyr-tubulin), MTs rich in Glu-tubulin were not found during early stages of development even by using an image intensification camera. Elevated levels of microtubular Glu-tubulin were first detected after CNS condensation in neurone processes. In addition, sperm tails, which remained remarkably stable inside the embryo until late stages of development, were decorated by ID5. This was in marked contrast to the distribution of microtubule arrays containing acetylated alpha-tubulin, which could already be detected during the cellular blastoderm stage. Additional experiments with taxol suggested that the absence of MTs rich in Glu-tubulin during early stages of development was not due to the rapid turnover rate of MTs, which would be too fast for alpha-tubulin to be detyrosinated. The possible significance of the differential detyrosination and acetylation of microtubules during development is discussed.

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum.

The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. The mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cytoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.