Eric S. Cole

A β-Tubulin Mutation Selectively Uncouples Nuclear Division and Cytokinesis in Tetrahymena thermophila

ABSTRACT 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 β-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.

Developmental progression of Tetrahymena through the cell cycle and conjugation.

The ciliate Tetrahymena thermophila can be said to undergo a variety of developmental programs. During vegetative growth, cells coordinate a variety of cell-cycle operations including macronuclear DNA synthesis and a-mitotic fission, micronuclear DNA synthesis and mitosis, cytokinesis and an elaborate program of cortical morphogenesis that replicates the cortical organelles. When starved, cells undergo oral replacement, transformation into fast-swimming dispersal forms or, when encountering cells of a complementary mating type, conjugation. Conjugation involves a 12 hour program of meiosis, mitosis, nuclear exchange and karyogamy, and two postzygotic divisions of the fertilization nucleus. This chapter reviews experimental data exploring the developmental dependencies associated with both vegetative and conjugal development.

A Proteomics Approach to Cloning Fenestrin from the Nuclear Exchange Junction of Tetrahymena

ABSTRACT. We set out to find the “fenestrin” gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila. First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS‐PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP‐localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.

Nested Genes CDA12 and CDA13 Encode Proteins Associated with Membrane Trafficking in the Ciliate Tetrahymena thermophila

ABSTRACT We describe a novel pair of nested genes, CDA12 and CDA13, from Tetrahymena thermophila. Both are implicated in membrane trafficking associated with cell division and conjugation. Green fluorescent protein localization reveals Cda12p decoration of diverse membrane-bound compartments, including mobile, subcortical tubulovesicular compartments; perinuclear vesicles; and candidates for recycling endosomes. Cda13p decorates intracellular foci located adjacent to cortically aligned mitochondria and their neighboring Golgi networks. The expression of antisense CDA12 RNA in transformants produces defects in cytokinesis, macronuclear segregation, and the processing of pinosomes to downstream compartments. Antisense CDA13 RNA expression produces a conjugation phenotype, resulting in the failure of mating pairs to separate, as well as failures in postconjugation cytokinesis and macronuclear fission. This study offers insight into the membrane trafficking events linking endosome and Golgi network activities, cytokinesis, and karyokinesis and the unique membrane-remodeling events that accompany conjugation in the ciliate T. thermophila. We also highlight an unusual aspect of genome organization in Tetrahymena, namely, the existence of nested, antisense genes.

A Cytogenetic Study of Development In Mechanically Disrupted Pairs of Tetrahymena Thermophila

ABSTRACT We examined the nuclear behavior of mating Tetrahymena cells that had been mechanically disrupted at various times throughout conjugation. Disruption was achieved by agitating conjugating Tetrahymena in the presence of 0.1‐3 mm glass beads. Two minutes of agitation with 1 mm beads yielded optimal pair disruption (70%) with high viability (92%). Disrupting pairs between 0‐4.7 h after the initiation of mating produced mostly disrupted conjugants in which development was aborted. However, as many as 20% of these early disrupted conjugants completed development even without their mating partners. After 5 h the percentage of disrupted conjugants completing development increased dramatically, reaching 80% by 6.7 h. These results support a model suggesting that events associated with nuclear exchange and fusion 5 h into conjugation trigger a commitment to completion of the postzygotic developmental program. the early conjugants that completed development following disruption suggest that development can be sustained even in the absence of a mating partner. This represents a novel method of bringing the micronuclear genome into macronuclear expression with minimal cytoplasmic exchange between partners. We discuss these results in light of a model relating cortical and nuclear signaling events that reciprocally drive conjugal development.

The Tetrahymena Conjugation Junction

Those who study ciliates have struggled over the years to establish a place in the pantheon of model organisms.1 Eukaryotic cell biologists have turned profitably to yeast models for the powerful genetic tools at their disposal, while developmental biologists have cultivated a gallery of metazoan embryos with contributions from the plant and fungal worlds. Yet ciliates continue to contribute to fundamental aspects of both cell biology and development, often by extreme example, and among the ciliates, Tetrahymena has emerged as one of the stars.

The Role of Cortical Geometry in the Nuclear Development of Tetrahymena thermophila

Abstract Vegetative cells were subjected to electrofusion and the resulting heteropolar doublets were then mated to normal single cells and followed throughout conjugation using cytological and genetic techniques. The unique cyto-geometry created in a heteropolar doublet—a continuous cytoplasmic compartment bounded by two anterior poles and sharing a fused posterior pole at midbody, and the potential for two conjugal exchange junctions—resulted in instructive perturbations of nuclear behavior. Our results indicate that the course of nuclear development is strongly dependent on the cortical geometry of conjugating cells. Specifically, 1) continuation of development after meiosis requires an established conjugal junction; 2) after pronuclear exchange, pronuclei are subjected to attractive forces; and 3) products of the second postzygotic division are actively positioned near the posterior region of the cell cortex where they develop into micronuclei.

Abnormal Micronuclear Telomeres Lead to an Unusual Cell Cycle Checkpoint and Defects in Tetrahymena Oral Morphogenesis

ABSTRACT Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cells oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.

Membrane Dynamics at the Nuclear Exchange Junction during Early Mating (One to Four Hours) in the Ciliate Tetrahymena thermophila

ABSTRACT Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.

Development in Electrofused Conjugants of Tetrahymena thermophila

Abstract Electric shock can create parabiotic fusions of living Tetrahymena cells. In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs. In particular, we electrofused pairs from normal [diploid × diploid] matings with vegetatively dividing cells in G- or M-phase of the cell cycle. We also fused [diploid × diploid] conjugants with mating pairs involving an aneuploid partner [diploid × “star”], which typically undergo an abortive conjugal pathway termed genomic exclusion. Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: 1) the “abort” signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the “terminal commitment point”, a developmental stage in normal [diploid × diploid] pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division). Finally we demonstrate that a conjugal-arrest-activity varies with the vegetative cell cycle, reaching its highest level of activity during M-phase and dropping just after cytokinesis.