Rapid changes in tubulin RNA synthesis and stability induced by deflagellation in Chlamydomonas

Detachment of the flagella of Chlamydomonas induces a rapid accumulation of mRNAs for tubulin and other flagellar proteins. Measurement of the rate of alpha and beta tubulin RNA synthesis during flagellar regeneration shows that deflagellation elicits a rapid, 4-7- fold burst in tubulin RNA synthesis. The synthesis rate peaks within 10- 15 min, then declines back to the predeflagellation rate. Redeflagellation of cells at times before the first flagellar regeneration is completed (and when cells have already accumulated elevated levels of tubulin RNA) induces another burst in tubulin RNA synthesis which is identical to the first in magnitude and duration. This finding indicates that the induction signal may act to simply reprogram the tubulin genes for a transient burst of maximal synthesis. Evidence is presented that the stability of the tubulin RNAs changes during regeneration. Stability changes include both an apparent stabilization during regeneration and accelerated decay following regeneration.     

Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15–20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth is 80–85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella $\text{1}\text{3}\text{–}\text{1}\text{2}$ normal length. Nevertheless, in vivo labeling with ¹⁴C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction of tubulin synthesis.Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.     

A conditional cell division mutant of Chlamydomonas reinhardii having an increased level of colchicine resistance

Conditional cell division mutants were isolated from Chlamydomonas reinhardii. They were unable to form colonies at 34 °C but not at 23 °C. One of the mutants, TS-60, could neither divide at high nor at low (15 °C) temperature, and seemed to continue protein synthesis at restrictive temperatures. TS-60 also exhibited resistance to 6 mM colchicine which inhibited cell division of the wild-type. Observing that TS-60 flagella were highly resistant to colchicine in their regeneration, it is concluded that the mutational alteration has affected not only the mitotic apparatus but also the flagella. Thermolability of TS-60 was not detected in flagellation but in cell division, though colchicine resistance was expressed in both flagellation and cell division. This suggests that the stable formation of the flagellar microtubule mainly depends on the specific organization of its component. Both thermolability and colchicine resistance of TS-60 were inherited in a Mendelian fashion and unseparable from each other. Reversion tests indicated that the two characters were caused by a single mutation. It is inferred that the above-mentioned phenotypes of TS-60 are the consequence of a mutation in factor(s) involving the colchicine binding activity of tubulin and that this mutational change pleiotrophically leads to some impediment in microtubule formation at restrictive temperature.     

Temperature-shock induction of multiple flagella induces additional synthesis of flagellum specific mRNAs and tubulin

Four mRNAs (alpha- and beta-tubulin, flagellar calmodulin and Class-I), specifically expressed when Naegleria amebae differentiate into flagellates, were followed at 5-10 min intervals during the temperature-shock induction of multiple flagella in order to better understand how basal body and flagellum number are regulated. Surprisingly, tubulin synthesis continued during the 37 min temperature shock. An initial rapid decline in alpha- and beta-tubulin and flagellar calmodulin mRNAs was followed by a rapid re-accumulation of mRNAs before the temperature was lowered. mRNA levels continued to increase until they exceeded control levels by 4-21%. Temperature shock delayed flagella formation 37 min, produced twice as much tubulin protein synthesis and three fold more flagella. Labeling with an antibody against Naegleria centrin suggested that basal body formation was also delayed 30-40 min. An extended temperature shock demonstrated that lowering the temperature was not required for return of mRNAs to near control levels suggesting that induction of multiple flagella and the formation of flagella per se are affected in different ways. We suggest that temperature-shock induction of multiple flagella reflects increased mRNA accumulation combined with interference with the regulation of the recently reported microtubule-nucleating complex needed for basal body formation.     

Matrix interferences in the analysis of benzene in urine

The analysis of benzene in urine of the general population or of exposed workers can be performed with different methods using the ‘purge and trap’ or ‘solid-phase microextraction’ techniques in combination with gas chromatographic analysis and photoionisation or mass spectrometric detection. The published results, however, are deeply conflicting. Differences in sample preparation by different research groups and our own preliminary observations prompted us to investigate pre-analytical and analytical factors potentially capable of modifying the urinary benzene quantification results. Benzene concentrations were measured in 20 urine samples in relation to different conditioning conditions (at 24, 40 and 80°C) and at basic or acid pH. Urinary protein concentrations were measured in the same samples. Urine heating at 80°C yields benzene concentrations on average five times higher than at 24°C. On acidification of urine, the benzene released increases up to 28-fold in comparison to that obtained at uncorrected ‘physiological’ pH. Despite a widely scattered data distribution, a statistically significant linear correlation was found between ‘heat-released’ and ‘acid-labile’ benzene values. There was no correlation between total urinary proteins present in ‘physiological’ concentrations (between 12 and 110 mg/l) and the different kinds of benzene in urine. Our results could perhaps be explained if it is supposed that part of the benzene in urine is absorbed onto sediment, or bound to specific proteins, or derived from parent molecules and is released with pH modification or heat administration. Our observations may also help to explain why the urinary benzene concentrations reported by different investigators vary considerably even when environmental levels are comparable.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5°C, multiplying at 33°C) and cold-sensitive (arrested at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in ‘gently lysed cells’ (obtained by treatment with 0.01% Brij-58) or ‘highly lysed cells’ (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [³H]dTTP efficiently, while highly lysed cells exhibited a low level of [³H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extracts reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.     

A temperature-sensitive N-glycosylation mutant of S. cerevisiae that behaves like a cell-cycle mutant

The temperature-sensitive S. cerevisiae mutant alg1-1, defective in the N-glycosylation of proteins, shows a first cycle arrest at the non-permissive temperature of 36 °C. The cell number increases by 50% and the absorbance approximately doubles. The budding index of 0.4 at 26 °C drops to 0.15 and DNA synthesis quickly comes to a halt at 36 °C. When the temperature is lowered again, budding and DNA synthesis start after a lag of 2–3 h; α-factor prevents both these processes in cells of mating type a. In addition, cells arrested at 26 °C in G1 with α-factor also do not start budding at the non-permissive temperature after removal of α-factor. The results support recent findings obtained with tunicamycin and suggest that at least one glycoprotein is required for G1-S phase transition in yeast.     

How is the flagellar length of mature sperm determined? : II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 μm, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 μm, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.     

Thermotropic ‘two-stage’ liquid crystalline crystalline lipid phase separation in microsomal membranes

The effect of temperature on native microsomal membrane vesicles isolated from Tetrahymena is investigated by wide angle X-ray diffraction. A 4.2 Å reflection, typical for lipids in the crystalline state, can be recorded in the temperature range between 0°C and 35°C. Quantitative evaluation of this reflection reveals a broad thermotropic ‘two-stage’ liquid crystallinecrystalline lipid phase separation with a ‘breakpoint’ at approx. 18°C. This ‘breakpoint’ coincides with the emergence of lipid-protein segregations in endomembranes of intact Tetrahymena cells as previously visualized by freeze-etch electron microscopy.     

The influence of the herbicide trifluralin on flagellar regeneration in Chlamydomonas

The mode of action of trifluralin is known to include disruption of cell division in root meristems by causing an absence of spindle microtubules. It has also been shown that trifluralin binds to tubulin isolated and purified from Chlamydomonas flagella. In this paper the kinetics of in vivo flagellar regeneration was used as a model to determine the influence of trifluralin on tubulin assembly. Chlamydomonas cells were grown in synchronous culture using a 12 h light-dark cycle. At 3 h into the light cycle the cells were subjected to shear force to induce flagellar abortion. Flagellar regeneration, in the presence of varying concentrations of trifluralin, was observed by Nomarski interference microscopy. After 1.5 h, trifluralin concentrations below 0.1 μM had not affected the regeneration rate, while concentrations above 5 μM prevented the onset of regeneration. As the concentration between 0.1 and 5 μM was increased, the final length of all flagella decreased. Using combinations of cycloheximide and trifluralin it was determined that trifluralin did not influence tubulin synthesis, and removing trifluralin only restored 50% of the regeneration capacity present at the beginning of treatment. By comparing groups of cells where the tubulin pool was depleted or present, it was found that trifluralin prevented assembly rather than causing a breakdown of previously assembled flagella. The research reported here supports the theory that the mechanism of action of trifluralin is an interaction of trifluralin and tubulin in a way that prevents tubulin assembly into spindle microtubules.     

FLAGELLAR DEVELOPMENT AND REGENERATION IN VOLVOX CARTERI (CHLOROPHYTA)1

The biflagellate somatic cells of Volvox carteri f. nagariensis lyengar exhibit an asymmetric pattern of flagellar development. Initiallt each somatic cell has two short (4 μm) flagella but after several hours one flagellum on each cell elongates unitl it reaches a length of 12 μm. Due to the regular arrangement of somatic cells in the Volvox spheroid it is apparent that the same flagellum on each somatic is the first to elongale. The asymmetric flagellar length is maintained for about 8 h after which the second flagellum on each somatic cell elongates. When the second flagellum attains the same length (12 μm) as the first flagellum, both flagella elongale at the same rate until reaching a final length of 22 μm. Experimental removal of somatic cell flagella results in their regeneration. Somatis cells regenerate both flagella simultaneously and full length flagella are produced in about 2 h. The intial rate of flagellar regeneration is about ten times faster than the intial rate of flagllar growth in development. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial rate of flagellar regeneration but the flagella produced in the presence of the drug are half the length of flagella produced in its absence. Somatic cells are able to regenerate flagella up to the time of α and β tubulin, the major structural proteins of the flagellar axoneme, and other cellular proteins.     

Delay ripening of ‘Qingnai’ plum (Prunus salicina Lindl.) with 1-methylcyclopropene

‘Qingnai’ plum fruit were treated with 0, 250, 500 or 1000 nL L⁻¹ of 1-methylcyclopropene (1-MCP) for 6 h and stored at 20 °C. The fruit firmness, peel color, chlorophyll content, titratable acidity (TA), respiration rate and ethylene production, chlorophyllase, pectin methylesterase (PME) and polygalacturonase (PG) activities were monitored during postharvest ripening of ‘Qingnai’ plums. ‘Qingnai’ plums without 1-MCP treatment soften very rapidly at room temperature after harvest, showing a continuing decrease in hue angle, chlorophyll content, TA and increase in chlorophyllase, PME and PG activities during postharvest storage. In contrast, the 1-MCP-treated fruits showed reduced ethylene production and respiration rate and delayed softening, which was associated with the reduction in the activity of PME and PG. The 1-MCP treatment also significantly inhibited the chlorophyllase activity and peel color development in ‘Qingnai’ plums during postharvest ripening at 20 °C. These results suggest that 1-MCP treatment may be useful for maintaining the fruit quality and extending the postharvest shelf-life of ‘Qingnai’ plums.     

Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii

A series of conditional mutants of the algal, biflagellate Chlamydomonas reinhardtii with temperature-sensitive defects in flagellar assembly and function were isolated. The genetics and phenotypes of 21 mutants displaying a rapid alteration in flagellar function upon shift from the permissive (20 degrees C) to the restrictive (32 degrees C) temperatures are described. These mutants designated as "drop-down" or dd-mutants have been placed in four categories on the basis of their defective phenotypes: (a) dd-assembly mutants - the preformed flagella are resorbed at 32 degrees C and reassembly of flagella is inhibited; (b) dd-fragile flagella mutants - the flagella are lost by detachment at 32 degrees C, but can be reassembled; (c) dd-motility mutants - the flagella are retained at 32 degrees C, but are functionally defective; (d) dd-lethal mutants - display combined defects in flagellar function and cell growth. Tetrad analysis of the mutants back-crossed to wild-type, recombination analysis of intermutant crosses, and complementation tests in the construction of heterozygous diploid strains indicate that at least 14 nuclear genetic loci are represented among 21 mutants. The availability of temperature-sensitive mutations affecting the assembly and function of the flagellum suggests that the morphogenesis of this complex eukaryotic organelle is amenable to genetic dissection.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

Adaptation to different growth temperatures modifies some mammalian cell survival responses

Chinese hamster (HA-1) cells that have been grown at 37 °C since explant several years ago can adapt themselves to grow at temperatures ranging from 32 to 41 °C. This growth adaptation is accompanied by major phenotypic changes in, for exampie, the cellular responses to 43 and 45 °C heat challenges and to ethanol challenges (0–10% in concentration). Cells grown at 39.5 °C are seen to acquire substantial heat resistance when compared with cells grown at 37 °C; resistance is even more pronounced if the growth temperature is at 41 °C. On the other hand, cells grown at 32 °C become more sensitive to heat than controls. Our results also indicate an increased resistance to ethanol of the 41 °C grown cells. By contrast the cells' X-ray survival response is affected only minimally. The changes seen are phenotypic; upon being returned to 37 °C, HA-1 cells within 34 h regain their ‘normal’ heat responses.     

Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.     

Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.     

Flagellar regeneration in the scaly green flagellateTetraselmis striata (Prasinophyceae): regeneration kinetics and effect of inhibitors

Flagellar regeneration after experimental amputation was studied in synchronized axenic cultures of the scaly green flagellateTetraselmis striata (Prasinophyceae). After removal of flagella by mechanical shearing, 95% of the cells regrow all four flagella (incl. the scaly covering) to nearly full length with a linear velocity of 50 nm/min under standard conditions. Flagellar regeneration is independent of photosynthesis (no effect of DCMU; the same regeneration rate in the light or in the dark), but depends on de novo protein synthesis: cycloheximide at a low concentration (0.35 μM) blocks flagellar regeneration reversibly. No pool of flagellar precursors appears to be present throughout the flagellated phase of the cell cycle. A transient pool of flagellar precursors, sufficient to generate 2.5 μm of flagellar length, however, develops during flagellar regeneration. Tunicamycin (2 μg/ml) inhibits flagellar regeneration only after a second flagellar amputation, when flagella reach only one third the length of the control. Flagellar regeneration inT. striata differs considerably from that ofChlamydomonas reinhardtii and represents an excellent model system for the study of synchronous Golgi apparatus (GA) activation, and transport and exocytosis of GA-derived macromolecules (scales).