Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively. Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential. A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin. In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.     

DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila.

We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila. DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation. Anlagen DNA was first detectably methylated at GATC sites 3-5 hours after its synthesis. Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation. The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells. Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time. Upon pair separation, less than 20% of old macronuclear DNA remained. A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing anlagen.     

Caspase-like activity is required for programmed nuclear elimination during conjugation in Tetrahymena

During conjugation in the binucleate ciliate, Tetrahymena thermophila, the old macronucleus is eliminated as new macronuclei and micronuclei are ontogenetically derived from the zygote nucleus. The mechanism of programmed nuclear elimination in ciliates may be related to the mechanism of apoptosis in higher organisms since its chromatin undergoes major condensation, its DNA is digested into nucleosome-sized fragments, and it stains positively for TUNEL. The present study explores whether caspases are involved in programmed macronuclear degradation in Tetrahymena. We show here that caspase-like activity is detectable using two specific colorimetric substrates, and that the activity is reduced with specific caspase inhibitors. In addition, using the fluorigenic substrate PhiPhiLux, active caspase-like activity is detected in living cells, localized to cytoplasmic vesicles; activity is not detected in pre- or post-condensed macronuclei. Finally, three different inhibitors of caspase activity cause a block to macronuclear chromatin condensation and elimination. Therefore, a caspase-like enzyme activity is necessary for regulating macronuclear elimination in Tetrahymena. These data support the possibility that macronuclear elimination is related, evolutionarily, to regulated cell death in multicellular organisms.     

Caspase-like activity in programmed nuclear death during conjugation of Tetrahymena thermophila

Apoptosis, or programmed cell death, is common in a variety of eucaryotes, from unicellular protozoa to vertebrates. The ciliated protozoan Tetrahymena thermophila has a unique apoptosis-like nuclear death during conjugation, called programmed nuclear death. This death program involves nuclear condensation (pyknosis) and oligonucleosomal DNA fragmentation in the parental macronucleus. Subsequently, the condensed nucleus is entirely resorbed in the autophagosome. Here we demonstrate that caspase-8- and -9-like activity was detected, but no caspase-3-like activity, by in vitro assay during the nuclear resorption process, suggesting that caspase-like activity is associated with both programmed cell death and apoptosis-like nuclear death in Tetrahymena. The use of indicator dye to detect the loss of mitochondrial membrane potential suggested the uptake of mitochondria and the degenerating macronucleus by the autophagosome. An involvement of mitochondria in the programmed nuclear death is discussed.     

The CNA1 histone of the ciliate Tetrahymena thermophila is essential for chromosome segregation in the germline micronucleus

Ciliated protozoans present several features of chromosome segregation that are unique among eukaryotes, including their maintenance of two nuclei: a germline micronucleus, which undergoes conventional mitosis and meiosis, and a somatic macronucleus that divides by an amitotic process. To study ciliate chromosome segregation, we have identified the centromeric histone gene in the Tetrahymena thermophila genome (CNA1). CNA1p specifically localizes to peripheral centromeres in the micronucleus but is absent in the macronucleus during vegetative growth. During meiotic prophase of the micronucleus, when chromosomes are stretched to twice the length of the cell, CNA1p is found localized in punctate spots throughout the length of the chromosomes. As conjugation proceeds, CNA1p appears initially diffuse, but quickly reverts to discrete dots in those nuclei destined to become micronuclei, whereas it remains diffuse and is gradually lost in developing macronuclei. In progeny of germline CNA1 knockouts, we see no defects in macronuclear division or viability of the progeny cells immediately following the knockout. However, within a few divisions, progeny show abnormal mitotic segregation of their micronucleus, with most cells eventually losing their micronucleus entirely. This study reveals a strong dependence of the germline micronucleus on centromeric histones for proper chromosome segregation.     

The transition from conjugal development to the first vegetative cell division is dependent on RAD51 expression in the ciliate Tetrahymena thermophila

Rad51p, the eukaryotic homolog of the prokaryotic recA protein, catalyzes strand exchange between single- and double-stranded DNA and is involved in both genetic recombination and double-strand break repair in the ciliate Tetrahymena thermophila. We have previously shown that disruption of the Tetrahymena RAD51 somatic macronuclear locus leads to defective germline micronuclear division and that conjugation of two somatic rad51 null strains results in an early meiotic arrest. We have constructed Tetrahymena strains that are capable of RAD51 expression from their parental macronuclei and are homozygous, rad51 nulls in their germline micronuclei. These rad51 null heterokaryons complete all of the early and middle stages of conjugation, including meiosis, haploid nuclear exchange, zygotic fusion, and the programmed chromosome fragmentations, sequence eliminations, and rDNA amplification that occur during macronuclear development. However, the rad51 null progeny fail to initiate the first vegetative cell division following conjugal development. Coincident with the developmental arrest is a disproportionate amplification of rDNA, despite the maintenance of normal total DNA content in the developing macronuclei. Fusion of arrested rad51 null exconjugants to wild-type cells is sufficient to overcome the arrest. Cells rescued by cytoplasmic fusion continue to divide, eventually recapitulating the micronuclear mitotic defects described previously for rad51 somatic nulls.     

Role of histone deacetylation in developmentally programmed DNA rearrangements in Tetrahymena thermophila

In Tetrahymena, as in other ciliates, development of the somatic macronucleus during conjugation involves extensive and reproducible rearrangements of the germ line genome, including chromosome fragmentation and excision of internal eliminated sequences (IESs). The molecular mechanisms controlling these events are poorly understood. To investigate the role that histone acetylation may play in the regulation of these processes, we treated Tetrahymena cells during conjugation with the histone deacetylase inhibitor trichostatin A (TSA). We show that TSA treatment induces developmental arrests in the early stages of conjugation but does not significantly affect the progression of conjugation once the mitotic divisions of the zygotic nucleus have occurred. Progeny produced from TSA-treated cells were examined for effects on IES excision and chromosome breakage. We found that TSA treatment caused partial inhibition of excision of five out of the six IESs analyzed but did not affect chromosome breakage at four different sites. TSA treatment greatly delayed in some cells and inhibited in most the excision events in the developing macronucleus. It also led to loss of the specialized subnuclear localization of the chromodomain protein Pdd1p that is normally associated with DNA elimination. We propose a model in which underacetylated nucleosomes mark germ line-limited sequences for excision.     

External GTP binding and induction of cell division in starved Tetrahymena thermophila

The extracellular nucleotide, guanosine 5'-triphosphate (GTP) is known to be a chemorepellent for ciliated protozoa such as Paramecium and Tetrahymena. Here, we studied the surface localization of GTP binding sites and also its effects on the cell division of Tetrahymena thermophila. When a ribose-modified and fluorescent analog of GTP, 2'-(or -3')-O-trinitrophenyl (TNP)-GTP was added to the cells starved in non-nutrient buffer, a remarkable fluorescence was observed at the compound cilia of the oral area, while it was weak at other cilia and the somatic membrane. Following transfer of the cells to the starvation medium, the intensity of TNP-GTP fluorescence at the oral area gradually increased and was saturated at 3-4 hours. Addition of GTP to the starved cell induced not only an avoiding reaction in swimming, but also induced a synchronous cell division that occurred 2 hours later. An attempt to search for other stimuli, which induced cell division, revealed that mechanical stimulation by a short period of centrifugation was almost as effective as the addition of GTP. The supernatant after centrifugation had the ability to induce cell division, and such activity was abolished after the supernatant was treated with the phosphatase, apyrase, suggesting the release of GTP by the mechanical stimulation. These results indicate that the released GTP binds mainly to the oral area and this then induces the cell division of starved T. thermophila.     

Diverse sequences within Tlr elements target programmed DNA elimination in Tetrahymena thermophila

Tlr elements are a novel family of ~30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleus-retained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ~23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA.     

Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.     

An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila

Programmed DNA rearrangements are important processes present in many organisms. In the ciliated protozoan Tetrahymena thermophila, DNA rearrangements occur during the sexual conjugation process and lead to the deletion of thousands of specific DNA segments and fragmentation of the chromosomes. In this study, we found that the Ku80 homologue, a conserved component of the nonhomologous end-joining process of DNA repair, was essential for these two processes. During conjugation, TKU80 was highly expressed and localized to the new macronucleus, where DNA rearrangements occur. Homokaryon TKU80-knockout mutants are unable to complete conjugation and produce progeny and are arrested at the two-micronuclei/two-macronuclei stage. Analysis of their DNA revealed failure to complete DNA deletion. However, the DNA-cutting step appeared to have occurred, as evidenced by the presence of circularized excised DNA. Moreover, chromosome breakage or de novo telomere addition was affected. The mutant appears to accumulate free DNA ends detectable by terminal deoxynucleotidyl transferase dUTP nick end labeling assays that led to the degradation of most DNA in the developing macronucleus. These findings suggest that Tku80p may serve an end-protective role after DNA cleavage has occurred. Unexpectedly, the large heterochromatin structures that normally associate with DNA rearrangements failed to form without TKU80. Together the results suggest multiple roles for Tku80p and indicate that a Ku-dependent DNA-repair pathway is involved in programmed DNA rearrangements in Tetrahymena.     

Locus-dependent profiles of the rescue of nonexcitable behavioral mutants during conjugation in Tetrahymena thermophila

The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes. When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about l h after pair formation. The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2h, they were not distinguishable from the wild-type homotypic pairs. On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs. When tnrA was mated with tnrB more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect. Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca²⁺ channels. © 1992 Wiley-Liss, Inc.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: Early expression of the zygotic genotype

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1–2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macro-nuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. © 1992 Wiley-Liss, Inc.     

Requirements for DNA replication preceding cell division in Tetrahymena pyriformis

Populations of Tetrahymena pyriformis were synchronized by 30 min heat shocks at 34 °C separated by 160 min intervals at the normal growth temperature. The cells initiate DNA synthesis immediately after the cellular division, and the S period of the population lasts about 80 min. It was found that DNA replication is a prerequisite for the following synchronous division. Inhibition of the DNA synthesis in early S by starvation of the cells for thymidine prevents the forthcoming division. However, inhibition in the latter half of S does not prevent the subsequent division. Thus the cells have synthesized enough DNA to permit cell division before the end of a normal S period. These results are discussed in relation to the organization of the genome replication in the highly polyploid macronucleus.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.