Determination of division plane and organization of contractile ring in Tetrahymena

In the molecular mechanism of division plane determination and contractile ring formation, Tetrahymena 85kDa protein (p85) is localized to the presumptive division plane before the formation of the contractile ring. p85 directly interacts with Tetrahymena calmodulin (CaM) in a Ca2+-dependent manner, and p85 and CaM colocalize in the division furrow. A Ca2+/CaM inhibitor N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI (W7) inhibits the direct interaction between p85 and Ca2+/CaM. W7 also inhibits the localization of p85 and CaM to the division plane, and the formation of the contractile ring and division furrow. In addition, p85 binds to G-actin in a Ca2+/CaM dependent manner, but does not bind F-actin. Tetrahymena profilin is localized to division furrow and binds Tetrahymena elongation factor-1alpha (EF-1alpha). EF-1alpha, which induces bundling of Tetrahymena F-actin, is also localized to the division furrow during cytokinesis. The evidence also indicates that Ca2+/CaM inhibits the F-actin-bundling activity of EF-1alpha, and that EF-1alpha and CaM colocalize in the division furrow. In this review, we propose that the Ca2+/CaM signal and its target protein p85 cooperatively regulate the determination of the division plane and the initiation of the contractile ring formation, and that profilin and a Ca2+/CaM-sensitive actin-bundling protein, EF-1alpha, play pivotal roles in regulating the organization of the contractile ring microfilaments.     

Localization of microtubules during macronuclear division in Tetrahymena and possible involvement of macronuclear microtubules in 'amitotic' chromatin distribution

The ciliated protozoa Tetrahymena contains two nuclei, a micronucleus and a macronucleus. In the vegetatively growing cell, the macronucleus divides amitotic while the micronucleus divides by mitosis. It has been indicated that microtubules are involved in macronuclear division and microtubules are observed to exist in the dividing macronucleus. To clarify the localization and the organization of microtubules in the amitotic dividing macronuclei, we used immunofluorescent staining technique. The microtubules were observed in the cytoplasm and macronucleus. The microtubules were organized and dynamically changed their distribution throughout the macronuclear division. We suggest a possibility that these microtubules are involved in 'amitotic' distribution of chromatin throughout the macronuclear division.     

p85 binds to G-actin in a Ca(2+)/calmodulin-dependent manner, thus regulating the initiation of cytokinesis in tetrahymena

Tetrahymena p85 is localized to the presumptive division plane before the formation of contractile ring microfilaments. p85 binds to calmodulin in a Ca(2+)-dependent manner and both proteins colocalize to the division furrow. Inhibition of the binding of p85 and Ca(2+)/calmodulin prevents both the localization of p85 and calmodulin to the division plane and the formation of the contractile ring, suggesting that the interaction of p85 and Ca(2+)/calmodulin is important in the formation of the contractile ring. We investigated the mechanisms of the formation of contractile ring, and the relationship among p85, CaM, and actin using co-sedimentation assay: p85 binds to G-actin in a Ca(2+)/calmodulin-dependent manner, but does not bind to F-actin. Therefore, we propose that a Ca(2+)/calmodulin signal and its target protein p85 are cooperatively involved in the recruitment of G-actin to the division plane and the formation of the contractile ring.     

Tetrahymena elongation factor-1 alpha is localized with calmodulin in the division furrow

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to ribosomes. We previously reported that Tetrahymena EF-1 alpha induced the formation of bundles of rabbit skeletal muscle filamentous actin (F-actin) as well as Tetrahymena F-actin [Kurasawa et al. (1996) Zool. Sci. (Tokyo) 13, 371-375], and that Ca(2+)/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1 alpha [Kurasawa et al. (1996) J. Biochem. 119, 791-798]. In the present study, we investigated the binding between Tetrahymena EF-1 alpha and CaM using a Tetrahymena EF-1 alpha affinity column, and the localization of EF-1 alpha and CaM by indirect immunofluorescence. Only CaM in the Tetrahymena cell extract bound to Tetrahymena EF-1 alpha in a Ca(2+)-dependent manner. In interphase Tetrahymena cells, EF-1 alpha and CaM are colocalized in the crescent structure of the oral apparatus and the apical ring, while in dividing cells, they are colocalized in the division furrow. This is the first report describing the coexistence of EF-1 alpha and CaM in the division furrow, suggesting that EF-1 alpha and CaM are involved in the organization of contractile ring microfilaments during cytokinesis.     

The Nuclear Apparatus of the Ditransversal Ciliate Homalozoon vermiculare (Ciliophora,Rhabdophora) during Interphase and Division. I. The Macronucleus1

ABSTRACT. The nuclear apparatus of Homalozoon vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. The number of macronuclear segments depends (i) on the number of divisions of individual segments during the interphase and (ii) on the number of segments that arise prior to cytokinesis from the (temporary) filiform macronucleus. Precytokinetic changes of the macronucleus involve the fusion of individual segments followed by contraction and subsequent elongation of the entire macronucleus. The chromatin bodies uncoil into fine fibrils during macronuclear contraction. At the time when the division furrow appears, the macronucleus starts to renodulate. The interphase segment contains a more or less reticulated chromatin body partly attached to the nuclear envelope and about 30 polymorphous nucleoli. The latter consist of the pars granulosa, the pars fibrosa, and an additional fibrillar component. The nucleoli undergo drastic changes prior to division and the granular component disappears completely during macronuclear condensation. On the average, the macronucleus contains a 3,400-fold amount of DNA compared with a haploid micronucleus, but the intraspecific differences in the DNA content of the entire macronucleus are extremely large. In contrast, DNA content and size of an individual segment of the macronucleus are precisely regulated during interphase.     

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila

The ciliated protozoan Tetrahymena thermophila contains two distinct nuclei within a single cell-the mitotic micronucleus and the amitotic macronucleus. Although microtubules are required for proper division of both nuclei, macronuclear chromosomes lack centromeres and the role of microtubules in macronuclear division has not been established. Here we describe nuclear division defects in cells expressing a mutant beta-tubulin allele that confers hypersensitivity to the microtubule-stabilizing drug paclitaxel. Macronuclear division is profoundly affected by the btu1-1 (K350M) mutation, producing cells with widely variable DNA contents, including cells that lack macronuclei entirely. Protein expressed by the btu1-1 allele is dominant over wild-type protein expressed by the BTU2 locus. Normal macronuclear division is restored when the btu1-1 allele is inactivated by targeted disruption or expressed as a truncated protein. Immunofluorescence studies reveal elongated microtubular structures that surround macronuclei that fail to migrate to the cleavage furrows. In contrast, other cytoplasmic microtubule-dependent processes, such as cytokinesis, cortical patterning, and oral apparatus assembly, appear to be unaffected in the mutant. Micronuclear division is also perturbed in the K350M mutant, producing nuclei with elongated early-anaphase spindle configurations that persist well after the initiation of cytokinesis. The K350M mutation affects tubulin dynamics, as the macronuclear division defect is exacerbated by three treatments that promote microtubule polymerization: (i) elevated temperatures, (ii) sublethal concentrations of paclitaxel, and (iii) high concentrations of dimethyl sulfoxide. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with 3-methyladenine or wortmannin also induces amacronucleate cell formation in a btu1-1-dependent manner. Conversely, the myosin light chain kinase inhibitor ML-7 has no effect on nuclear division in the btu1-1 mutant strain. These findings provide new insights into microtubule dynamics and link the evolutionarily conserved PI 3-kinase signaling pathway to nuclear migration and/or division in Tetrahymena.     

Reorganization of microtubules in the amitotically dividing macronucleus of tetrahymena

We developed a modified immunofluorescence protocol that permitted visualization of microtubules inside the macronucleus of the ciliate Tetrahymena. Although the amitotically dividing macronucleus lacks a spindle, an elaborate system of microtubules is assembled inside the macronucleus and between the macronucleus and the cortex. Microtubules could not be detected inside the interphase macronuclei. The early stage of macronuclear division was associated with the assembly of short macronuclear microtubules that localized randomly. The intramacronuclear microtubules were subsequently organized in a radial manner. During elongation of the macronucleus, the distribution of macronuclear microtubules changed from radial to parallel. During constriction of the macronucleus, dense and tangled macronuclear microtubules were detected at the region of nuclear constriction. In the cytosol, microtubules were linking the macronucleus and cell cortex. During recovery after drug-induced depolymerization, microtubules reassembled at multiple foci inside the macronucleus in close proximity to the chromatin. We propose that these microtubules play roles in chromatin partitioning, macronuclear constriction, and positioning of the macronucleus in relation to the cell cortex.     

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.     

TIF1 activates the intra-S-phase checkpoint response in the diploid micronucleus and amitotic polyploid macronucleus of Tetrahymena

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.     

Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis

The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.     

Homologues of radial spoke head proteins interact with Ca2+/calmodulin in Tetrahymena cilia

Calmodulin (CaM) is an axonemal component. To examine the pathway of Ca(2+)/CaM signaling in cilia, using Ca(2+)/CaM-affinity column, we identified seven Ca(2+)/CaM-associated proteins from a crude dynein fraction and isolated 62 kDa (p62) and 66 kDa (p66) Ca(2+)/CaM-associated proteins in Tetrahymena cilia. The amino acid sequences deduced from the p62 and p66 cDNA sequences suggested that these proteins were similar to Chlamydomonas radial spoke proteins 4 and 6 (RSP4 and RSP6), components of the radial spoke head, and sea urchin sperm p63, which is a homologue of RSP4/6, and isolated as a key component that affect flagellar bending patterns. Although p62 and p66 do not have a conventional CaM-binding site, those have consecutive sequences which showed high normalized scores (>or= 5) from a CaM target database. These consecutive sequences were also found in RSP4, RSP6, and p63. These radial spoke heads proteins have a high similarity region composed of 15 amino acids between the five proteins. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized along the outer edge of the curved central pair microtubules in axoneme. Therefore, it is possible that the interaction between Ca(2+)/CaM and radial spoke head control axonemal curvature in the ciliary and flagellar waveform.     

Roles of three domains of Tetrahymena eEF1A in bundling F-actin

The conventional role of eukaryotic elongation factor 1A (eEF1A) is to transport aminoacyl tRNA to the A site of ribosomes during the peptide elongation phase of protein synthesis. eEF1A also is involved in regulating the dynamics of microtubules and actin filaments in cytoplasm. In Tetrahymena, eEF1A forms homodimers and bundles F-actin. Ca(2+)/calmodulin (CaM) causes reversion of the eEF1A dimer to the monomer, which loosens F-actin bundling, and then Ca(2+)/CaM/eEF1A monomer complexes dissociate from F-actin. eEF1A consists of three domains in all eukaryotic species, but the individual roles of the Tetrahymena eEF1A domains in bundling F-actin are unknown. In this study, we investigated the interaction of each domain with F-actin, recombinant Tetrahymena CaM, and eEF1A itself in vitro, using three glutathione-S-transferase-domain fusion proteins (GST-dm1, -2, and -3). We found that only GST-dm3 bound to F-actin and influences dimer formation, but that all three domains bound to Tetrahymena CaM in a Ca(2+)-dependent manner. The critical Ca(2+) concentration for binding among three domains of eEF1A and CaM were < or =100 nM for domain 1, 100 nM to 1 microM for domain 3, and >1 microM for domain 2, whereas stimulation of and subsequent Ca(2+) influx through Ca(2+) channels raise the cellular Ca(2+) concentration from the basal level of approximately 100 nM to approximately 10 microM, suggesting that domain 3 has a pivotal role in Ca(2+)/CaM regulation of eEF1A.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.     

Evidence for Chromosomal Macronuclear Substructures in Tetrahymena*†

SYNOPSIS.
Under the growth conditions employed, the G₁ macronucleus of Tetrahymena pyriformis HSM contains 7.4 × 10^-12 g DNA, the G₂ micronucleus 0.42 × 10^-12 g. DNA content from the Tetrahymena thermophila macronucleus did not significantly differ from that of HSM, but the micronucleus contained about twice as much DNA as the micronucleus of the HSM cells. The T. thermophila macronucleus contained on average enough DNA for ˜ 35 haploid micronuclear copies. A new spreading technic allowed separation of macronuclear substructures from cells of late G₂ to early G₁. Photometric determination of DNA content of 345 individual structures suggested the existence of 5 different-sized macronuclear structures with a DNA content corresponding to 2, 4, 8, and 16 × the basic values. Comparison of the DNA content of these structures with (a) mitotic micronuclear chromosomes and (b) meiotic micronuclear chromosomes of T. thermophila cells suggests that the 5 basic values of macronuclear structures derive from structures of micronuclear chromosomes. The micronuclear chromosomes of T. pyriformis may be oligotenic. It is suggested that these results further our understanding of macronuclear organization.     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

Nuclear Behavior of Euplotes woodruffi During Conjugation

SYNOPSIS. During conjugation of E. woodruffi , the micro-nucleus divides repeatedly four times prior to synkaryon formation and twice thereafter. The first division resembles an ordinary somatic mitosis, resulting in the formation of two daughter nuclei in each conjugant. Both products of this division enter the second division which corresponds to the heterotypic division of other ciliates, characterized by a parachute stage. Following this stage sixteen bivalents appear and separate into dyads and pass to the poles. During the following divisions individualized chromosomes do not appear but only certain chromatin elements comparable to those seen in the somatic and preliminary divisions. These divide and pass to the poles. All daughter nuclei of the second division enter and complete the third division. Only two of the products of the third division enter the final pregamic division while the rest degenerate. Exchange of pronuclei and their fusion leads to synkaryon formation. The conjugants then separate and in each exconjugant the synkaryon divides twice in rapid succession. Of the four products one condenses to become the functional micronucleus, another enlarges rapidly to become the macronuclear anlage while the remaining two degenerate and disintegrate. The old macronucleus breaks into irregular and polymorphic bodies. As the macronuclear anlage enlarges the remnants of the old macronucleus reorganize and fuse with the macronuclear anlage to form a characteristic vegetative macronucleus.