Joseph Bryan

Separation and interaction of the major components of sea urchin actin gel.

Abstract Warming of low calcium, isotonic extracts of sea urchin eggs causes gelation by inducing the polymerization of F-actin which then interacts with other proteins in the cytosol to form the gel. Two major protein components with molecular weights of 58,000 and 220,000 have been identified. F-actin has previously been purified from such preparations and shown to interact with a mixture of these proteins to form a gel composed of F-actin aggregates displaying a unique 11 nm banding pattern. The gelation is calcium-sensitive; half-maximal inhibition is at 1 to 2 μ m free calcium in the extract. Solubilization of the gel in 0.6 m -KI and chromatography on A-15m agarose in 0.6 m -KCI resolves the 220K protein (Mr = 220,000) from G-actin and the 58K protein (Mr = 58,000). Further purification on DEAE-cellulose yields pure 58K protein. When combined, the repolymerized actin and 58K protein form microscopic needles composed of linear aggregates of F-actin with the characteristic 11 nm banding. Saturation of the 58K bindingsites of F-actin occurs at ratios of one 58K protein/4 to 5 actin molecules. This actin—58K protein interaction does not show calcium sensitivity. Addition of the 220K protein to pure F-actin produces no detectable aggregation and this protein therefore appears to be distinct from the high molecular weight, actin-binding proteins isolated from vertebrates. The addition of the 220K protein to solutions of needles causes aggregation to a material morphologically indistinguishable from the original gel, but this gelation is not sensitive to calcium. These results show that the 58K protein links F-actin filaments to form cross-banded needles with a 11 nm periodicity, which are then linked into a three-dimensional gel by the 220K protein. Theso results suggest that the initial calcium sensitivity may be due to the presence of regulatory proteins which are removed during the purification.

Assembly of tubulin from cultured cells and comparison with the neurotubulin model.

Abstract Spontaneous microtubule assembly can be obtained in extracts from a variety of cultured cell lines by including glycerol in the assembly buffer. An analysis of the effects of cultured cell extracts on brain tubulin (neurotubulin) assembly has shown that the extracts contain initiation inhibitors whose effects are diminished by glycerol. By using glycerol during the assembly step, cultured cell tubulin can be purified by assembly-dissassembly procedures. The amount of glycerol necessary for significant spontaneous assembly varies from 1–6 M among the different cell lines and is dependent upon their content of inhibitor. Comparison of the assembly products obtained from NA, C 6 and CHO cells at increasing glycerol concentrations shows that glycerol enhances the purification of tubulin and a polypeptide of molecular weight 49,000 daltons in all three systems. These preparations contain a number of other polypeptides, including a group with gel electrophoretic mobilities characteristic of tau-factor, but lack the high molecular weight microtubule-associated proteins (MAPs) which are present in neurotubulin preparations. Phosphocellulose chromatography of NA tubulin removes several proteins from the tubulin and results in a loss of polymerizability. Among three proteins which are completely removed from the inactive tubulin, the most prominent is the 49K protein. This observation and the co-purification of the 49K protein with tubulin through two assembly-disassembly cycles suggest that it is a true MAP. The difference in MAP proteins between brain and tissue culture cells is parallelled by an absence of ring structures in NA tubulin preparations. NA tubulin, however, does form rings when brain MAPs are added. The early steps of NA tubulin assembly differ from those of neurotubulin; no rings are involved, and the first assembly intermediates are straight protofilament bundles. The differences between MAPs from cultured cells and brain and the absence of ring formation in NA tubulin preparations suggest that the assembly model based on neurotubulin is not completely general. A comparison of extracts from CHO cells grown with and without dibutyryl cAMP revealed no differences between the behavior of these extracts in spontaneous tubulin assembly or in mixture experiments with brain tubulin.

Studies on clonal cartilage strains: I. Effect of contaminant non-cartilage cells

Abstract Methods for the establishment and propagation of clonal chondrocyte strains were presented. Under certain conditions these clonal strains produced matrix for extended periods. The addition of a small number of non-cartilage cells to these strains resulted in a rapid loss of phenotype. The possible relation of this effect to the dedifferentiation observed in mass cultures is discussed.

Organization of myosin in a submembranous sheath in well-spread human fibroblasts☆

Abstract Using an indirect immunofluorescence technique, antibodies against human platelet myosin stain a highly organized layer of sheath of fibers in well-spread human fibroblasts. By adjusting the focus, the sheath is seen to be continuous over the top of the cell. Myosin staining along a fiber is periodic with a mean spacing of 0.71 ± 0.1 μm. Although the sheath becomes progressively disorganized and eventually disappears upon cell rounding after trypsinization, the mean spacing in the fibers remaining at intermediate stages does not appear to change. The organization is sensitive to cytochalasin B (CB), but insensitive to colchicine. Anti-myosin staining of the sheath persists in cells lysed in 0.1–0.5% Triton X-100 at low ionic strength despite some myosin extraction. Preparation of the cytoskeleton at increasingly higher ionic strength or extraction with ATP or pyrophosphate diminishes the sheath staining with anti-myosin. A detailed analysis of the mean myosin spacing in the cytoskeleton after various treatments designed to induce contraction suggests (1) that reagents which extract myosin, i.e., ATP, high salt and pyrophosphate, all cause contraction and (2) that the mean spacing does not detectably change. Anti-alpha-actinin and anti-tropomyosin also localize and demonstrate a periodicity for their respective antigens in the sheath fibers. Double staining with anti-myosin and anti-tropomyosin gives an enhancement of the basic myosin perodicity indicating co-localization. Double staining with anti-alpha-actinin and anti-myosin gives a nearly continuous fluorescence along the sheath fiber suggesting a complementary periodicity. This finding was confirmed with a direct anti-myosin-indirect anti-alpha-actinin staining experiment. A possible chemical mechanism, based on a competition of these proteins for actin, for the generation of the observed periodicity and formation of the sheath is considered.

Characterization of acetyl-3H-labeled vinblastine binding to vinblastine-tubulin crystals.

Purified vinblastine-tubulin crystals, induced in vivo by incubation of unfertilized sea urchin eggs with [acetyl-3H]vinblastine sulfate, contained 0·7 to 1·0 mole of bound vinblastine per mole of tubulin in a form which dissociated only when crystals were suspended in stabilizing buffer containing free vinblastine. Vinblastine was found to bind to crystals with a stoichiometry of two mole per mole of tubulin in vitro. Vinblastine bound to both sites with similar affinities of 2·4 × 105 liters per mole (18°C). The binding of vinblastine to crystals was not affected appreciably by temperature between 0°C and 35°C, and was not at all influenced by the presence of high concentrations of GTP, colchicine, griseofulvin, or calcium ions. Vinblastine binding to crystals of tubulin was competitively inhibited by other active vinca alkaloid derivatives, and the abilities of the derivatives to bind crystal tubulin paralleled the abilities of the derivatives to inhibit polymerization of brain microtubules in vitro.

PRELIMINARY STUDIES ON AFFINITY LABELING OF THE TUBULIN-COLCHICINE BINDING SITE*

The present results argue that irradiation of a colchicine-tubulin complex at 366 nm results in destruction of the binding site and the labeling of a small molecule soluble in chloroform-methanol.

Studies on clonal cartilage strains: II. Selective effects of different growth conditions

Abstract Growth conditions were described which vary the relative number of variant cells that accumulate. Maintenance of a high division rate through regular subculture minimizes the accumulation of variants. This results in a population which expresses the cartilage phenotype after many population doublings.