Amino-acid sequence data of beta-tubulin from Physarum polycephalum myxamoebae

Starting with 7.7 mg of a beta-tubulin isolated from myxamoebae of the slime mould Physarum polycephalum, 90% of the sequence has been determined by the Edman degradation of peptides generated by cyanogen bromide, trypsin and Staphylococcus aureus protease. Differences to other beta-tubulins are mainly conservative and spread evenly throughout the chain except for a high concentration at the C-terminus. The Physarum beta-tubulin shows most homology to Chlamydomonas beta-tubulin (90.5%) and least homology to yeast beta-tubulin (S. cerevisiae, 73.4%). Two tryptic peptides were isolated in approximately equal quantities which were identical except in one position (S/ALTVPELTQRMFDA) showing that at least two beta-tubulins are present in myxamoebae. However, since this was the only heterogeneity found, these beta-tubulins are probably very similar.     

Isolation and sequencing of alpha-tubulin peptides from myxamoebae of the slime mould Physarum polycephalum

Starting from only 5.9 mg of alpha-tubulin from myxamoebae of the slime mould Physarum polycephalum, we have isolated and sequenced peptides that account for 96% of the complete sequence. The peptides were generated by digestion of alpha-tubulin with trypsin, Staphylococcus aureus protease and cyanogen bromide. They were then separated according to size on a TSK G2000 SW column using a 10 mM ammonium acetate buffer at pH 6.8. In addition to good peptide separations, a time-consuming desalting step with subsequent loss of material was unnecessary because the relatively small amount of ammonium acetate could be removed by lyophilization. High resolution of peptides from the TSK fractions was achieved on C4 or C18 reverse-phase columns by eluting with a gradient of acetonitrile in 50 mM ammonium acetate (pH 6.8) and in 0.1% trifluoroacetic acid, respectively. The peptides were then sequenced using a gas phase sequencer.     

Membrane histochemistry of Physarum polycephalum myxamoebae

Myxamoebae of Physarum polycephalum are the uninucleate, haploid stage of the organism. Histochemical studies were undertaken to characterize intracellular and plasma membranes, and to provide a basis for assaying subcellular fractions for enrichment in plasma membranes. Lead salts deposition techniques were employed for hydrolytic enzymes. Alcian blue-ruthenium red, osmium tetroxide-potassium ferrocyanide, and phosphotungstic acid-chromic acid stains were evaluated for specificity for plasma membranes. Glucose 6-phosphatase was localized in endoplasmic reticulum, Golgi apparatus, and perinuclear space. 5'-Nucleotidase was localized in food vacuoles, chromatin, and plasmalemma. Acid phosphatase was in food vacuoles and Golgi apparatus. Alkaline phosphatase was in food vacuoles and endoplasmic reticulum. We conclude that none of the above enzymes is suitable as a cytochemical marker for plasma membranes of Physarum myxamoebae, but recommend instead staining ultrathin sections of membrane pellets with phosphotungstic acid-chromic acid, which stains plasma membranes selectively.     

A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis

As the first step towards correlating structure and function of tubulin in the slime mold Physarum polycephalum we have elucidated the nucleotide sequence of a cDNA that appears to code for all but the last 25 to 30 C-terminal amino acids of a plasmodial alpha-tubulin. Differences in amino acid sequence from those of other alpha-tubulins are distributed fairly evenly throughout the sequence, although a relatively extensive conserved region is found in position 396 to 426 near the C terminus. A small region in position 298 to 307 contains a cluster of amino acid residues unique to Physarum alpha-tubulin. The sequence is 70% homologous to two yeast alpha-tubulins and about 83% homologous to five animal alpha-tubulins. A comparison of the homologies of all the known alpha-tubulins indicates that a large decrease in the accepted point mutation rate has occurred during the evolution of the metazoa, suggesting a major functional specialization of microtubules.     

In vitro assembly of microtubule proteins from myxamoebae of Physarum polycephalum

Microtubule protein of >95% purity has been isolated by self-assembly from concentrated cell extracts of myxamoebae of Physarum polycephalum. Ninety-eight percent of the amoebal microtubule protein was tubulin. Both a and β subunits of amoebal tubulin were different from neurotubulin α and β subunits, but very similar to those of Tetrahymena ciliary tubulin. The non-tubulin components, which co-purified with tubulin through three assembly cycles, were essential to microtubule formation and contained several polypeptides including some of apparent molecular weights 49000, 57000 and 59000. Purified amoebal microtubule protein formed microtubules on warming in the absence of glycerol which were cold- and Ca²⁺-labile. In vitro, microtubule assembly was inhibited by vinblastine, benzimidazole derivatives and griseofulvin, but not by 10^?4 M colchicine. Amoebal tubulin had a much lower affinity than neurotubulin for colchicine.     

Distribution of acetylated alpha-tubulin in Physarum polycephalum

The expression and cytological distribution of acetylated alpha-tubulin was investigated in Physarum polycephalum. A monoclonal antibody specific for acetylated alpha-tubulin, 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094), was used to screen for this protein during three different stages of the Physarum life cycle--the amoeba, the flagellate, and the plasmodium. Western blots of two-dimensional gels of amoebal and flagellate proteins reveal that this antibody recognizes the alpha 3 tubulin isotype, which was previously shown to be formed by posttranslational modification (Green, L. L., and W. F. Dove, 1984, Mol. Cell. Biol., 4:1706-1711). Double-label immunofluorescence demonstrates that, in the flagellate, acetylated alpha-tubulin is localized in the flagella and flagellar cone. Similar experiments with amoebae interestingly reveal that only within the microtubule organizing center (MTOC) are there detectable amounts of acetylated alpha-tubulin. In contrast, the plasmodial stage gives no evidence for acetylated alpha-tubulin by Western blotting or by immunofluorescence.     

Identification and characterization of microtubule proteins from myxamoebae of Physarum polycephalum.

Cell extracts of myxamoebae of Physarum polycephalum have been prepared in such a way that they do not inhibit assembly of brain microtubule protein in vitro even at high extract-protein concentration. Co-polymers of these extracts and brain tubulin have been purified to constant stoichiometry and amoebal components identified by radiolabelling. Amoebal tubulin has been identified as having an alpha-subunit, mol.wt. 54 000, which co-migrates with brain alpha-tubulin and a beta-subunit, mol.wt. 50 000, which co-migrates with Tetrahymena ciliary beta-tubulin. Non-tubulin amoebal proteins that co-purify with tubulin during co-polymer formation have been shown to be essential for microtubule formation in the absence of glycerol and appear to be rather more effective than brain microtubule-associated proteins in stimulating assembly. The mitotic inhibitor griseofulvin (7-chloro-2',4,6-trimethoxy-6'-methylspiro[benzofuran-2(3H),1'-cyclohex-2'-ene] -3,4'-dione), which binds to brain microtubule-associated proteins and inhibits brain microtubule assembly in vitro, affected co-polymer microtubule protein in a similar way, but to a slightly greater extent.     

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

Flow cytometry of the differentiation of Physarum polycephalum myxamoebae to cysts

Myxamoebae of Physarum polycephalum, strain Cld, were grown on agar lawns on live bacteria. Myxamoebae were harvested, fixed and stained with propidium iodide. Flow cytometry showed that, as in the case of Physarum plasmodia, there is no G1 phase during rapid exponential growth. However, an apparent G1 phase was observed at the end of exponential growth when the culture arrested with the G1 DNA content for about a day between growth and differentiation. Most myxamoebae differentiated into cysts, but some formed microplasmodia and others appeared to lose DNA. The cysts possessed the G2 phase DNA content and there was an S phase connecting the G1-arrested state with the encysted state. Encystment was blocked by hydroxyurea (HU) suggesting that DNA synthesis is essential for encystment. The natural temporary synchronization in G1 phase may provide the basis of a method for selecting mutants with a conditional block in G2 or M phases.     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976     

Demonstration of different patterns of microtubule organization in Physarum polycephalum myxamoebae and plasmodia using immunofluorescence microscopy

We have used anti-tubulin antibodies and immunofluorescence microscopy to determine the overall distribution of microtubules during interphase and mitosis in both the myxamoebae and plasmodia of the slime mold Physarum polycephalum. We have paralleled these observations with electron microscopy of the same stages. The myxamoebae possess a network of cytoplasmic microtubules whilst the coenocytic plasmodium does not possess any cytoplasmic microtubules--at either interphase or mitosis. In plasmodia microtubules are, however, elaborated by an intranuclear microtubule organizing centre (MTOC) during prophase of mitosis and these microtubules proceed to form part of the mitotic spindle. There is little difference in the overall distribution and arrangement of microtubules during division of either the myxamoebal or plasmodial nuclei. These findings are discussed in relation to the synthesis of tubulin during the plasmodial cell cycle and the rearrangements of the nuclear envelope during mitosis.     

The heterogeneity of cytoplasmic deoxyribonucleic acid polymerase from Physarum polycephalum.

Cytoplasmic DNA polymerase (DNA deoxynucleotidyltransferase, EC 2.7.7.7) was partially purified from Physarum polycephalum. The first step of the purification procedure utilized the fact that the enzyme on gel filtration behaves in anomalous fashion. The second step was either ion-exchange chromatography or sucrose-density-gradient centrifugation. The partially purified DNA polymerase was heterogeneous and at least four species with different sedimentation coefficients (5.5S, 7.2S, 8.6S and 11.5S) were detected. Calculated molecular weights indicated a tendency for stoicheiometric polypeptide aggregation, accompanied by an alteration of the three-dimensional structure froma compact spheroid to a more open elliptical form. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and computed molecular weights suggest an active protomer in the range of 113000 daltons; all data pertain to I 0.045, which was maintained during the whole procedure.