Reply: whole-culture synchronization - effective tools for cell cycle studies

Studies of gene expression during the eukaryotic cell cycle in whole-culture synchronized cultures have been published using many methodologies. These procedures alter the state of the cell cycle for a population of cells, rather than purifying a population of cells that are in the same state. Criticism of these methods (e.g. see Cooper, this issue, pp. 266-269, ) suggests that these studies are flawed, and posits that such methodologies cannot be used to study the cell cycle because they alter the size and age distributions of the cultures. We believe that whole-culture cell cycle studies work even though they alter the size and age distributions: these cells still progress through the cell cycle and although we do not suggest that the methods are perfect, we will explain how these microarray studies have successfully identified cell cycle regulated genes and why these results are biologically meaningful.     

Revertants of a Chinese hamster ovary cell mutant with an altered beta-tubulin: evidence that the altered tubulin confers both colcemid resistance and temperature sensitivity on the cell

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20:29-36, 1980). This mutant carries an altered beta-tubulin but still grows normally at 37 degrees C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3 degrees C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered beta-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant beta-tubulin polypeptide. This second alteration appears to make the mutant beta-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in beta-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered beta-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.     

Ecto- or arbuscular mycorrhizas – which are best?

Ecological theories concerning the relationship of suites of traits with resource supply and plant potential growth rate have concentrated primarily on leaf structure and physiology – much less is known about interspecific patterns of variation in root structure and physiology. In this issue, Wahl & Ryser reduce this knowledge gap by demonstrating important correlations of root structure with potential growth rate and height among a range of perennial temperate grass species adapted to sites of a wide range of fertility (see pp. 459–471).     

Cover

Photoautotrophically grown Chlamydomonas reinhardtii CC1690 cells shifted from low to high light. The image was taken by M. Schroda with an Olympus BX53F Microscope using DIC optics. Chlamydomonas reinhardtii is the first microalga in which riboswitches have been applied to regulate transgene expression (see review by Nguyen et al. in this issue). [Vol. 52, No. 3, pp. 320–328]     

Tubulin is phosphorylated at tyrosine by pp60c-src in nerve growth cone membranes

We show here that tubulin is the major in vivo substrate of the tyrosine-specific protein kinase pp60c-src in nerve growth cone membranes. Phosphotyrosine antibodies were used to demonstrate phosphotyrosyl residues in a subpopulation of alpha- and beta-tubulin that was highly enriched in a subcellular fraction of growth cone membranes from fetal rat brain. The presence of phosphotyrosine- modified isoforms of alpha- and beta-tubulin in vivo was confirmed by 32p labeling of rat cortical neurons in culture. Tubulin in growth cone membranes was phosphorylated at tyrosine in endogenous membrane phosphorylation reactions (0.068 mol phosphotyrosine/mol alpha-tubulin and 0.045 mol phosphotyrosine/mol beta-tubulin), and phosphorylation was specifically inhibited by antibodies directed against pp60c-src, which is localized in the growth cone membranes. pp60c-src was capable of directly phosphorylating tubulin as shown in immune complex kinase assays with purified brain tubulin. Phosphopeptide mapping revealed a limited number of sites of tyrosine phosphorylation in alpha- and beta- tubulin, with similar phosphopeptides observed in vivo and in vitro. These results reveal a novel posttranslational modification of tubulin that could regulate microtubule dynamics at the growth cone.     

Dominant effects of tubulin overexpression in Saccharomyces cerevisiae.

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.     

Beta-tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas

Previous studies suggest that beta-tubulin isotype protein levels could be useful as indicators of nonsmall cell lung cancer (NSCLC) aggressiveness. However, measurement of protein amounts in tissue samples by staining techniques is semiquantitative at best. Since technologies for measuring mRNA levels have become more efficient and quantitative, we wanted to determine whether beta-tubulin message levels may be useful as biomarkers. Quantitative real-time RT-PCR was used to measure the seven classes of beta-tubulin isotypes, stathmin and MAP4 mRNA levels in 64 NSCLC and 12 normal lung tissue samples. We found significantly higher fractions of beta-tubulin classes II and V mRNA compared to the other isotypes in all lung tumor samples (P < 0.05). In addition, the ratio of beta-tubulin classes II/V mRNA was significantly higher in NSCLCs compared to normal lung tissues (P < 0.001). The data suggest that the ratio of beta-tubulin classes II and V mRNA could be useful as a biomarker for NSCLC tumor differentiation and/or NSCLC aggressiveness. Furthermore, the ratio of MAP4 to stathmin mRNA was found to be higher in diseased lung tissues compared to normal lung tissues, suggesting this ratio might also be used as a clinically relevant biomarker for NSCLCs.     

Application of stains-all staining to the analysis of axonemal tubulins: identification of beta-tubulin and beta-isotubulins.

The cationic dye, Stains-all, is known to stain brain beta-tubulin blue and alpha-tubulin red (Serrano, L. et al. (1986) J. Biochem. Biophys. Methods 12, 281-287; Serrano, L. et al. (1989) Biochem. Int., 19, 235-246). The present experiments show that this stain can also be applied to detect beta-tubulin in axonemal tubulins from various sources such as cilia of protozoa, sperm flagella of echinoderm, and sperm flagella of mollusc. Furthermore, these experiments showed that it selectively stains isoforms of axonemal beta-tubulin blue following isoelectric focusing, whereas those of alpha-tubulin are stained red. These results indicate that Stains-all staining is a useful tool for electrophoretic analysis of axonemal tubulins.     

Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin.

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.     

Sequence and expression of the chicken beta 5- and beta 4-tubulin genes define a pair of divergent beta-tubulins with complementary patterns of expression.

We have determined the nucleotide sequence of the chicken beta 5 (c beta 5)-tubulin gene. The gene displayed the coding structure common to all previously studied vertebrate beta-tubulin genes and was divided into four exon sequences interrupted by three intervening sequences (located between codons 19 and 20, within codon 56, and within codon 93). Comparison of the predicted polypeptide sequence encoded by c beta 5 with those of four other available chicken beta-tubulin sequences revealed that c beta 5 encoded a highly divergent beta-tubulin polypeptide isotype which was distinguished from previously known sequences primarily by two discrete variable sequence domains. However, c beta 5 uniquely shared identity in 16 residue positions with another divergent chicken beta-tubulin gene, c beta 4. These common sequences distinguished c beta 4 and c beta 5 from the remaining three chicken beta-tubulin genes. Analysis of the expression of c beta 5 and c beta 4 revealed a strikingly complementary pattern of gene expression: c beta 5 was expressed in a wide variety of cell and tissue types but not in neurons, whereas c beta 4 expression was detected uniquely in neuronal cells. Overall, these findings suggest the existence of two divergent families of beta-tubulin sequences in the chicken and further raise the possibility that the complementary expression of the c beta 4 and c beta 5 genes may fulfill a requirement for the presence of a divergent beta-tubulin polypeptide isotype in all cell types.     

Differential expression of two neural cell-specific beta-tubulin mRNAs during rat brain development.

We recently demonstrated that beta-tubulin mRNA expression is regulated during rat brain development. This is manifested by a dramatic decrease in both 1.8- and 2.9-kilobase (kb) mRNAs when extensive neurite elongation is occurring. Coincident with these decreases is the increased production of a 2.5-kb mRNA. (J.F. Bond and S.R. Farmer, Mol. Cell. Biol. 3:1333-1342, 1983). In the present study, we have isolated and characterized three different cDNAs corresponding to beta-tubulin mRNAs (R beta T.1, R beta T.2, and R beta T.3). Hybridization of 3' untranslated region subclones of R beta T.1 and R beta T.2 cDNAs to RNA from a variety of rat tissues and cells revealed that these two cDNAs are neural cell specific. R beta T.1 corresponds to an abundant 1.8-kb mRNA expressed only at early stages of rat brain development. R beta T.2 corresponds to the 2.5-kb mRNA expressed at later stages. These data strongly suggest that there is differential expression of the beta-tubulin multigene family during rat brain development.     

Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.     

Developmental regulation of calmodulin, actin, and tubulin RNAs during rat testis differentiation

Postnatal testis differentiation involves transition through neonatal, pre-meiotic, meiotic, haploid, and mature stages. We have examined the qualitative and quantitative changes in rat testis RNAs that specifically hybridize to cDNAs encoding the cytoskeletal proteins, calmodulin, beta-actin, alpha- and beta-tubulin at ages corresponding to each of these developmental periods. We compared the species and relative levels of specific RNAs from testes of animals engaged in normal spermatogenesis with RNA from germ cell-depleted, Sertoli cell-enriched (SCE) testis. Distinct developmental patterns of expression of the specific RNAs were found with each of the cDNAs in the two animal models. A 2.2 kb (kilobase) actin RNA and a 2.7 kb beta-tubulin RNA are maximal at 5-10 days of age, suggesting these RNAs are required by somatic and germ cells in the postnatal phase prior to puberty. Between 19 and 29 days, when pachytene spermatocytes appear in significant numbers, there is a slight increase in the 2.2-kb actin RNA, but a 4- to 10-fold increase in RNAs hybridizing to cDNAs for calmodulin, alpha- and beta-tubulin. These changes are much less pronounced in the SCE testis than in the normal testis, indicating increases in these RNAs are related to germinal cell maturation. The germ cell-related increase in 1.8-kb beta-tubulin RNA appears to reflect a developmental "switch" in the gene from which the RNA is derived. This hypothesis is based on the observation that the ratio of hybridization of a chicken brain beta-tubulin cDNA versus a rat spleen beta-tubulin cDNA to the 1.8-kb RNA band increases more than 40-fold between 5 and 29 days of age in normal testis, but is constant in SCE testis. These data suggest that a specific beta-tubulin gene is activated in maturing germ cells. Analogously, a 2.1-kb alpha-tubulin RNA is found only in maturing normal testis and increases as spermatids are produced. A 2.0-kb beta-tubulin RNA, not found in normal testes, is maximal in maturing SCE testes, suggesting this RNA is of somatic cell origin. All of the RNA species studied, except the 2.0-kb beta-tubulin RNA, decrease between 5 and 19 days in SCE testes, as Sertoli cell mitotic activity wanes, indicating that their levels may be regulated by the developmental signals that influence mitosis.     

Comparison of beta-tubulin mRNA and protein levels in 12 human cancer cell lines

Antimitotic drugs are chemotherapeutic agents that bind tubulin and microtubules. Resistance to these drugs is a major clinical problem. One hypothesis is that the cellular composition of tubulin isotypes may predict the sensitivity of a tumor to antimitotics. Reliable and sensitive methods for measuring tubulin isotype levels in cells and tissues are needed to address this hypothesis. Quantitative measurements of tubulin isotypes have frequently relied upon inferring protein amounts from mRNA levels. To determine whether this approach is justified, protein and mRNA levels of beta-tubulin isotypes from 12 human cancer cell lines were measured. This work focused on only beta-tubulin isotypes because we had readily available monoclonal antibodies for quantitative immunoblots. The percentage of beta-tubulin isotype classes I, II, III, and IVa + IVb mRNA and protein were compared. For beta-tubulin class I that comprises >50% of the beta-tubulin protein in 10 of the 12 cell lines, there was good agreement between mRNA and protein percentages. Agreement between mRNA and protein was also found for beta-tubulin class III. For beta-tubulin classes IVa + IVb, we observed higher protein levels compared to mRNA levels.Beta-tubulin class II protein was found in only four cell lines and in very low abundance. We conclude that quantitative Western blotting is a reliable method for measuring tubulin isotype levels in human cancer cell lines. Inferring protein amounts from mRNA levels should be done with caution, since the correspondence is not one-to-one for all tubulin isotypes.     

Changes in beta-tubulin isoforms and their RNA level in synchronized tobacco cells.

Tobacco BY-2 cells were synchronized by an aphidicolin treatment, and their beta-tubulin isoforms and their mRNA were analyzed by Western, Northern and dot blottings. The relative ratio of the beta-tubulin isoforms changed with the progress of cell cycle stage. By Northern blot hybridization of poly(A)+RNAs with a cloned carrot beta-tubulin cDNA probe, a single band of about 1.6 kb was detected throughout the cell cycle. Dot blot hybridization showed that beta-tubulin mRNA existed in all stages in the cell cycle at a relatively constant level, though it accumulated slightly more than average at M phase and decreased during G1 phase.     

Calcium binding properties of the beta tubulin subunit from chicken erythrocytes

Taxol-stabilised erythrocyte microtubules assembled less readily than similarly prepared brain microtubules on adding 10(-4) M-10(-3) M concentrations of calcium at 2 degrees C. Scatchard plot analyses of the high affinity calcium binding sites showed that the erythrocyte tubulin contained only 0.9 high affinity binding sites per dimer compared to 1.4 binding sites per dimer for brain tubulin. Association constants, however, for calcium binding to both erythrocyte and brain tubulin were similar (3.0 x 10(-6) M and 2.1 x 10(-6) M). The beta-tubulin subunit appeared to be responsible for the lower calcium binding ability of erythrocyte tubulin as shown by a gel overlay assay with 45Ca. Strains-all, a dye that stains many calcium binding proteins blue, did not stain erythrocyte beta-tubulin or its chymotryptic C-terminal fragment blue as was the case for brain beta-tubulin and its chymotryptic C-terminal fragment. We suggest that the lower calcium binding ability of erythrocyte beta-tubulin may be implicated in the differential behaviour of erythrocyte microtubules.