Mutual antagonism between Hippo signaling and cyclin E drives intracellular pattern formation.

Not much is known about how organelles organize into patterns. In ciliates, the cortical pattern is propagated during "tandem duplication," a cell division that remodels the parental cell into two daughter cells. A key step is the formation of the division boundary along the cell's equator. In Tetrahymena thermophila, the cdaA alleles prevent the formation of the division boundary. We find that the CDAA gene encodes a cyclin E that accumulates in the posterior cell half, concurrently with accumulation of CdaI, a Hippo/Mst kinase, in the anterior cell half. The division boundary forms between the margins of expression of CdaI and CdaA, which exclude each other from their own cortical domains. The activities of CdaA and CdaI must be balanced to initiate the division boundary and to position it along the cell's equator. CdaA and CdaI cooperate to position organelles near the new cell ends. Our data point to an intracellular positioning mechanism involving antagonistic Hippo signaling and cyclin E.

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

Two Antagonistic Hippo Signaling Circuits Set the Division Plane at the Medial Position in the Ciliate Tetrahymena.

In a single cell, ciliates maintain a complex pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes. The underlying molecular mechanisms of intracellular pattern formation in ciliates are largely unknown. Ciliates divide by tandem duplication, a process that remodels the parental cell into two daughters aligned head-to-tail. In the elo1-1 mutant of Tetrahymena thermophila, the segmentation boundary/division plane forms too close to the posterior end of the parental cell, producing a large anterior and a small posterior daughter cell, respectively. We show that ELO1 encodes a Lats/NDR kinase that marks the posterior segment of the cell cortex, where the division plane does not form in the wild-type. Elo1 acts independently of CdaI, a Hippo/Mst kinase that marks the anterior half of the parental cell, and whose loss shifts the division plane anteriorly. We propose that, in Tetrahymena, two antagonistic Hippo circuits focus the segmentation boundary/division plane at the equatorial position, by excluding divisional morphogenesis from the cortical areas that are too close to cell ends.

Cell context-specific effects of the beta-tubulin glycylation domain on assembly and size of microtubular organelles.

Tubulin glycylation is a posttranslational modification found in cells with cilia or flagella. The ciliate Tetrahymena has glycylation on ciliary and cortical microtubules. We showed previously that mutating three glycylation sites on beta-tubulin produces immotile 9 + 0 axonemes and inhibits cytokinesis. Here, we use an inducible glycylation domain mutation and epitope tagging to evaluate the potential of glycylation-deficient tubulin for assembly and maintenance of microtubular systems. In axonemes, the major defects, including lack of the central pair, occurred during assembly, and newly made cilia were abnormally short. The glycylation domain also was required for maintenance of the length of already assembled cilia. In contrast to the aberrant assembly of cilia, several types of cortical organelles showed an abnormally high number of microtubules in the same mutant cells. Thus, the consequences of deficiency in tubulin glycylation are organelle type specific and lead to either insufficient assembly (cilia) or excessive assembly (basal bodies and cortical microtubules). We suggest that the diverse functions of the beta-tubulin glycylation domain are executed by spatially restricted microtubule-associated proteins.

The Hippo Pathway Maintains the Equatorial Division Plane in the Ciliate Tetrahymena.

The mechanisms that govern pattern formation within the cell are poorly understood. Ciliates carry on their surface an elaborate pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes by largely unknown mechanisms. Ciliates divide by tandem duplication: the cortex of the predivision cell is remodeled into two similarly sized and complete daughters. In the conditional cdaI-1 mutant of Tetrahymena thermophila, the division plane migrates from its initially correct equatorial position toward the cell's anterior, resulting in unequal cell division, and defects in nuclear divisions and cytokinesis. We used comparative whole genome sequencing to identify the cause of cdaI-1 as a mutation in a Hippo/Mst kinase. CdaI is a cortical protein with a cell cycle-dependent, highly polarized localization. Early in cell division, CdaI marks the anterior half of the cell, and later concentrates at the posterior end of the emerging anterior daughter. Despite the strong association of CdaI with the new posterior cell end, the cdaI-1 mutation does not affect the patterning of the new posterior cortical organelles. We conclude that, in Tetrahymena, the Hippo pathway maintains an equatorial position of the fission zone, and, by this activity, specifies the relative dimensions of the anterior and posterior daughter cell.

A cyclin-dependent protein kinase homologue associated with the basal body domains in the ciliate Tetrahymena thermophila.

The tight coupling between cell cycle progression and morphogenetic development in the unicellular ciliates presents a unique model system for examination of the roles of Cdks in developmental processes. We here describe the isolation and characterization of the first cyclin-dependent kinase (Cdk) homologue, TtCdk1, from Tetrahymena thermophila. TtCdk1 corresponds to the larger of the two polypeptides recognized by anti-PSTAIRE antibody in a whole cell lysate, which differ from each other in their affinity for yeast p13(suc1) protein. In contrast to the constant protein expression levels of typical eukaryotic Cdks, the TtCdk1 protein level fluctuates periodically over the vegetative cell cycle, reaching a maximum at the end of the cell cycle, correlating with its histone H1 kinase activity. Its association with the membrane-skeletal domains that surround mature, but not nascent, basal bodies in the cell cortex suggests that TtCdk1 plays a role in the regulation of cortical morphogenesis in T. thermophila. A partial TtCDK1 knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei. Unlike the situations in higher eukaryotic cells, no apparent effect on basal body duplication was found upon disruption of the TtCDK1 gene.

Effect of alteration in the global body plan on the deployment of morphogenesis-related protein epitopes labeled by the monoclonal antibody 12G9 in Tetrahymena thermophila.

We have employed monoclonal antibodies to reinvestigate the janus mutants of the ciliate Tetrahymena thermophila, which cause reversal of circumferential polarity on the dorsal surface of the cell. This reversal brings about frequent ectopic expression of ventral cortical landmarks, such as a "secondary" oral apparatus, on the dorsal surface. The principal antibody employed, FXXXIX-12G9, immunolabels both transient cortical structures not directly associated with basal bodies (the fission line and the postoral meridional filament) and more permanent structures (apical band and oral crescent) that are associated with basal bodies. 12G9-immunolabeling of janus cells has revealed additional phenotypes, including disorder of ciliary rows. Further, this labeling has shown that the postoral meridional filament is often expressed and the apical band is frequently interrupted on the mid-dorsal surface of janus cells irrespective of whether or not these cells express a "secondary" oral apparatus. Of the permanent structures revealed by 12G9 immunofluorescence, modifications of the oral crescent (OC) are associated with prior modifications in the development of basal body-containing structures in the secondary oral apparatus. The formation of the apical band (AB) is also commonly abnormal in janus cells; analysis of specific abnormalities shows that the AB depends both on its initiation at a specific site near the anterior basal body of apical basal body couplets and on the normal location of these couplets just posterior to the fission line. We also have uncovered an intriguing difference in the reactivity of apical-band filaments to the 12G9 antibody in the two non-allelic janus mutants (janA1 and janC2) that we have investigated. Taken together, our observations indicate that the formation of new cellular structures at division depends both upon pre-existing cytoskeletal structures and upon the positional information provided by large-scale cellular polarities.

Morphogenesis: a Mob rules from the rear.

The giant ciliated protozoan Stentor coeruleus is re-emerging as a model organism for morphogenesis and patterning in unicellular organisms. A new study provides evidence that cytokinesis and morphogenesis are mechanistically linked through the Mob1 protein.

DisAp-dependent striated fiber elongation is required to organize ciliary arrays.

Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp's similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays.

From molecules to morphology: cellular organization of Tetrahymena thermophila.

Tetrahymena thermophila is both a cell and an organism, which combines great intracellular complexity with a remarkable accessibility to investigation using many different approaches. In this review, we start with a description of the elaborate cortical organization of the Tetrahymena cell, and then proceed inward to consider the mitochondria and then the nuclei. For each of these cellular organelles and organelle-systems, first we familiarize the reader with its location in the cell and its structure and ultrastructure, and then we analyze the molecular mechanisms associated with organelle assembly, function, and subdivision. This analysis includes a molecular inventory of the organelle or organelle system, as well as a review of the consequences of modification, disruption or overexpression of important molecular components of each structure or system. Relevant comparisons to results obtained with other well-studied organisms, from Paramecium to Homo sapiens, are also included. Our goal is to provide investigators, in particular those who are new to this organism, both the background and the motivation to work with this model system and achieve further insight into its organization and dynamics.

The actin gene ACT1 is required for phagocytosis, motility, and cell separation of Tetrahymena thermophila.

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Delta mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Delta cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Delta cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila.

A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.

Interactions between janus and bcd cortical pattern mutants in Tetrahymena thermophila : An investigation of intracellular patterning mechanisms using double-mutant analysis.

An analysis of bcd, janA; bcd, janB; and bcd, janC double-mutant phenotypes in Tetrahymena thermophila has allowed us to examine patterning processes affected by two different classes of mutations. bcd brings about a broadening of the oral and contractile vacuole pore domains in the ciliate cortex, while the janus mutations generate a mirror-image duplication of the ventral cortical pattern. We observed both bcd and janus characteristics expressed in the double mutants, as well as features unique to the double-mutant. Temperature-shift experiments employing the temperature-sensitive janB mutation in a double-mutant (bcd, janB) combination allowed us to observe the changes in pattern as a mirror-image geometry was brought into expression and subsequently removed within the bcd, janB double homozygote. These experiments suggest that there are multiple pattern-mechanisms at work with differing kinetics of expression in the ciliate cortex. We discuss how the bcd mutation could influance expression of the janus mutations in light of a model previously proposed to account for the janus phenotype.

Selective mirror-image reversal of ciliary patterns inTetrahymena thermophila homozygous for ajanus mutation.

The development of the oral apparatus (OA) and of neighboring ciliary structures ofTetrahymena thermophila was analyzed in cells homozygous for ajanus (jan A) mutation plus a recessiveenhancer of janA (eja). Such cells frequently possess two OAs located on opposite sides of the cell, a primary (1°) OA previously reported to be normal, and a secondary (2°) OA previously reported to express a mirror-reversal of right-left asymmetry. This study confirms the reality of a reversal in the gross orientation of membranelles in most developing 2° OAs. It also shows that there is a reversal of asymmetry in the pattern of resorption of basal bodies of ciliary rows adjacent to the 2° OA, and in the arrangement of basal-body couplets making up the portion of the apical "crown" of the cell situated close to the 2° OA. However, the locations at which membranelles of the 2° OA become modified during late phases of oral development remain normal, so that membranelles of 2° OAs are superimposable on those of 1° OAs. In addition, the membranelles of 2° OAs frequently undergo a rotation during the final phases of oral development, so that even their spatial orientation becomes normal. This mixture of reversed and normal features can be accounted for by postulating a superimposition of a reversed largescale asymmetry on a normal local asymmetry of ciliary units. This postulate predicts that no single mutation can bring about a complete mirror-image reversal of ciliary patterns.1° OAs appear normal by light microscopy. However, detailed analysis of SEM, preparations of isolated 1° OAs indicate subtle abnormalities of basal body arrangement in some of these OAs.