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R W Rubin

Publications and source records attributed to R W Rubin.

44 records · Page 3Linked to original sources
PubMed1974

Cyclic changes in the cell surface. II. The effect of cytochalasin B on the surface morphology of synchronized Chinese hamster ovary cells.

The surface morphology of Chinese hamster ovary cells treated with cytochalasin B (CB) has been examined using the scanning electron microscope. The cells respond to treatment with CB by retracting peripheral processes, rounding up, and assuming a smooth or gently convoluted surface. This response occurs within minutes. Cells in different stages of the cell cycle all respond in a similar manner. When CB is removed from treated cells by washing with conditioned medium, the cells regain their normal surface conformation within minutes. The surface topography of these released cells is characteristic of their stage in the cell cycle. Because CB causes an alteration in the morphology of the cell surface and because of the speed of the response and recovery, it is proposed that the primary site of action of CB is the cell surface.

Animals↗
PubMed1974

Effects of phosphotungstate negative staining on the morphology of the isolated Golgi apparatus.

Isolated Golgi complexes can be recognized in phosphotungstate (PTA) negative stain as stacks of membranous plates surrounded by a complex anastomosing network of tubules and vesicles. The extent of this tubular network is, however, much greater than can be observed in thin sections of whole cells. To determine which of the steps leading to the final negatively stained image may produce the observed changes, we have monitored each of the steps by other electron microscope and biochemical methods. The first damage to the membranes seems to occur during the initial isolation procedure as judged by the appearance of smooth patches on the freeze-fractured membrane faces that are normally covered with particles. Subsequent suspension of the Golgi fraction in water, to dilute the sucrose for negative staining, leads to the disappearnce of the stacking, to some tubulation and some vesiculation of the membranes as judged by thin section and freeze-cleave microscopy. The latter technique also reveals an increase in smooth-cleaving membrane faces. Application of the negative stain to the water-washed Golgi fraction, finally, produces extensive tubular arrays and a simultaneous decrease in the remaining large membranous vesicles. The freeze-cleaved tubular membranes appear essentially smooth except for small patches of aggregated particles. Parallel gel electrophoresis studies of the membranes and of the water and negative stain wash extracts indicate that protein extraction is involved in these morphological changes. PTA seems to be a particularly effective solvent for certain membrane proteins that are not removed by the water wash. These observations suggest that removal of membrane proteins alters structural restraints on the membrane lipids so that they behave semiautonomously like myelinics and form new artificial structures. This does not eliminate the possibility, however, that some tubules also exist in the Golgi apparatus in vivo.

Animals↗
PubMed1973

Adenosine 3',5'-cyclic monophosphate in Chlamydomonas reinhardtii. Influence on flagellar function and regeneration.

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.

Aminophylline↗
PubMed1973

Partial purification and phosphotungstate solubilization of basal bodies and kinetodesmal fibers from Tetrahymena pyriformis.

Previously devised methods for the isolation of basal bodies from ciliate protozoans were found to be inadequate for chemical analysis. We have modified and expanded these procedures and developed a method which gives preparations containing mainly basal bodies and kinetodesmal fibers. This procedure involved fixation of cells in 30% ETOH followed by digitonin or Triton X-100 solubilization and homogenization with a Brinkmann Polytron. This is followed by sucrose gradient centrifugation. Negative staining and thin sectioning revealed these preparations to be substantially more pure than those of previous workers. It was also found that neutralized phosphotungstate (PTA) solubilized many of the components present in fixed Tetrahymena. Neutralized 1.0% PTA solubilized axonemes, cortical, axonemal, and basal body microtubules as well as kinetodesmal fibers. These results have been confirmed by both electron microscope observations and gel electrophoresis of 100,000 g supernatants of the PTA extracts. A solution of 0.1% PTA did not affect the fibers but did solubilize basal bodies. Running 1.0% PTA extracts from our basal body fractions on sodium dodecyl sulfate (SDS) polyacrylamide gels allowed us to tentatively identify the peptides of basal bodies and kinetodesmal fibers. The latter structures appear to consist of a single 21,000 mol wt peptide. These results also suggest that great caution should be taken in interpreting PTA images, especially of microtubules and axonemes.

Animals↗
PubMed1973

The effect of cell-to-cell contact on the surface morphology of Chinese hamster ovary cells.

In the previous report (Porter et al., in this issue) morphological changes in Chinese hamster ovary (CHO) cells during the cell cycle were described. In this report we describe the role of intercellular contact on these changes. We find that intercellular contact is required for cells to exhibit the morphologies Porter et al. described for S and G(2). When cells are synchronized by mitotic selection and plated onto cover slips at very low density such that no intercellular contact occurs, the cells remain in a G(1) configuration (rounded and highly blebbed through G(1), S, and G(2)). This G(1) morphology is also observed in nonsynchronized log phase cells plated at low densities and allowed to grow for several generations. The addition of conditioned medium from confluent cultures does not induce low density cells to change morphology during the cell cycle. These results indicate that extensive intercellular contact is required for the complete expression of the morphological changes associated with the cell cycle (as described by Porter et al.). It is concluded that although classic contact inhibition of movement and of growth may be absent in this transformed cell line, some contact-dependent response persists.

Animals↗
PubMed1972

Alteration of nucleoside transport of Chinese hamster cells by dibutyryl adenosine 3':5'-cyclic monophosphate.

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N(6),O(2')-dibutyryl adenosine 3':5'-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [(3)H]-thymidine or [(3)H]uridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. [(3)H]Deoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [(3)H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 1 degrees was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 muM. At 37 degrees , the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acid-soluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP.

Aminophylline↗