Biomedical subjects
Publications and source records attributed to B M Jockusch.
A replica plating method for isolating it amoebal mutants of Physarum polycephalum has been devised. Temperature-sensitive mutations occur at a frequency after nitrosoguanidine mutagenesis of 10(-3) per survivor, are stable but are not usually expressed in the plasmodia formed from these amoebae in clones. Some of these mutants appear to be cell-cycle stage specific.
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The plasmodial stage of Physarum polycephalum contains up to 10(9) nuclei which undergo a naturally synchronous mitosis every 8 h. Nuclear processes such as DNA and RNA synthesis as well as many cytoplasmic processes such as histone synthesis are also synchronous. Physarum polycephalum is therefore widely used in studies of cell-cycle events. This article describes experiments that may help to explain two fundamental biological processes: (1) the mechanism that triggers mitosis, (2) the structural basis of mitotic movement.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
A protein was extracted from isolated nuclei of the slime mold Physarum polycephalum which could be labeled with radioactive precursors only during G(2) phase. The native protein was purified by extraction in low-ionic-strength buffer [10 mm tris(hydroxymethyl)aminomethane-hydrochloride] of isolated nuclei and by preparative polyacrylamide gel electrophoresis. It was extracted from isolated nucleoli. Its electrophoretic properties in three different polyacrylamide gel systems, its molecular weight (44,000 +/- 3,000), its precipitability by vincaleucoblastine, a vinca alkaloid, and its aggregation properties suggested that it might be actin. In a direct comparison with slime mold actin purified from the cytoplasm, no difference could be found between the two proteins in all these characteristics. The synthesis of cytoplasmic actin was not found to occur exclusively during G(2) phase. This suggested that nuclear actin was either synthesized independently from cytoplasmic actin or transported into the nuclei exclusively during G(2) phase. The possible role of nuclear actin during intranuclear mitosis is discussed.
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The size distribution and synthesis of polypeptide chains and the polysome patterns were studied during sporulation of the slime mold Physarum polycephalum, and were compared with nonsporulating controls. The proteins were divided into a 27,000 x g supernatant (buffer-soluble proteins) and a pellet (buffer-insoluble proteins) while still native. The sodium dodecyl sulfate complexes of the denatured proteins were separated on polyacrylamide gels containing urea. The following differences were found between sporulating and nonsporulating cultures. (i) The distribution of the soluble proteins into bands from sporulating and control cultures was the same in stained patterns; however, there was a slight shift toward increased synthesis of larger polypeptide chains in the radioactivity patterns of the soluble proteins in sporulating cultures. (ii) The amount of histones in the sporulating cultures was less than 30% of the values in the controls. Also, histone synthesis was reduced to less than 10% of that in the nonsporulating controls. In addition, proteins in three defined regions, corresponding to molecular weights of 70,000 to 75,000 (I), 55,000 (II), and 41,000 (III), were synthesized in sporulating cultures at a rate at least twice that in controls. Polypeptides corresponding to peaks I and II could be extracted from purified walls of mature spores. (iii) The polysome pattern as revealed by sucrose density centrifugation showed a breakdown of heavy polysomes at 3 hr after illumination, with their reappearance 4 hr later. The latter pattern, however, differed from that of the nonsporulating control in that the amount of light polysomes was reduced. This might account for the reduction in histone synthesis.
We have used high-resolution electron spectroscopic imaging to study the ultrastructure of negatively stained chicken gizzard vinculin. A careful examination of uranium salt-stained molecules revealed a high versatility of the overall shape of vinculin, for which an element of high flexibility is mainly responsible. This neck region links the vinculin head, probably consisting of the biochemically defined 90-kDa N-terminal fragment, to the rod-like tail. The hinge allows for sharp kinks in the molecule, so that head and tail structures can contact each other. By electron spectroscopic imaging, we were able to reveal substructural components in both head and tail regions. The head resembles a cloverleaf-like structure, consisting of three globular centers of mass, surrounding a protein-deficient center in a planar arrangement and of a short, stem-like fragment. The tail contains four spherical protein masses arranged like pearls on a string. Our data reveal a substructural organization of vinculin which is consistent with its presumed function as a structural component of microfilament attachment sites and support the concept of cryptic ligand-binding domains, previously based on biochemical evidence.
Capping proteins are F-actin binding proteins which interfere with the in vitro growth of an actin filament by blocking one of its ends (for recent reviews see refs 1-3). The majority of such proteins described so far "cap' the fast-growing (positive) end of the polar filament, thus reducing the velocity of filament growth while increasing the number of filaments being formed de novo from a monomer pool. We have studied the effects of capping proteins on the organization of actin filaments in living tissue culture cells by microinjection in conjunction with fluorescence, reflection contrast and electron microscopy. Our results, reported here, indicate that capping proteins from different sources disrupt microfilament bundles in a variety of cell types causing their disintegration from the distal end towards the centre of the cell.