Jason Wolfe

Differential Staining of Apoptotic Nuclei in Living Cells: Application to Macronuclear Elimination in Tetrahymena

Acridine orange (AO) has been used as a vital fluorescent stain to identify apoptotic cells in Drosophila, but little is known about what structures are stained. We explored the specificity of AO staining while studying nuclear apoptosis in Tetrahymena. Using AO alone or together with the vital nuclear stain Hoechst 33342 (HO), we find that lysosomes are generally clustered around the degenerating nucleus and that such nuclei are stained an orange-red color, like lysosomes. Significantly, the combined dyes, more so than with AO alone, distinguish between apoptotic and normal (or necrotic) nuclei by a clear color difference. Moreover, these dyes differentially stain apoptotic and normal nuclei in avian chondrocytes. The differential staining results are nullified in fixed cells or in cytoskeletal preparations treated with RNAse. Similarly, lysosomotrophic agents eliminate the differential staining. Our results are consistent with acidification of the apoptotic nucleus, possibly by fusion with lysosomes. However, even under basic conditions, the macronucleus condenses and is eliminated, suggesting that, if the nucleus is becoming acidified, acidification by itself is not essential for nuclear elimination. The differential staining procedure may provide a useful method for specifically identifying apoptotic cells and separating them for further analysis. (J Histochem Cytochem 45:675–683, 1997)

A cytological study of micronuclear elongation during conjugation in Tetrahymena.

Micronuclear elongation is the first major event in a series of nuclear changes occurring during the sexual stage of the life cycle of Tetrahymena. Beginning at about one hour after cells of complementary mating types have conjugated, the micronucleus leaves its recess in the macronucleus and swells slightly. This is accompanied by a reorganization of its chromatin from a reticular to a solid body. In the next stage the micronucleus assumes an egg shape, a development concomitant with the appearance of microtubules. While the chromatin “spins out” from the dense body, and microtubules increase in number, the nucleus assumes a spindle shape. During the elongation, which increases the length of the nucleus some fifty fold, microtubules are prominent in clusters just internal to the nuclear membrane, and parallel to the longitudinal axis of the nucleus. When elongation is completed the nucleus is curved around the macronucleus. Internally, partially condensed strands of chromatin are located off-center, towards the macronuclear side, and the density of the microtubules is diminished. At all the stages, DNA is located throughout the nucleus; neither discrete chromosomes nor synaptonemal complexes are seen. Occasionally cytoplasmic membrane systems are seen fused to the nuclear envelope which retains the typical appearance of a double membrane with pores.

Conjugation in Tetrahymena: The relationship between the division cycle and cell pairing☆

Abstract Conjugation, a sexual stage in the life cycle of Tetrahymena, is marked by the pairing of two cells of opposite mating types. Pairing establishes cytoplasmic continuity between the two cells and initiates the complex of nuclear events involved in sexual exchange. After mixing cells of opposite mating types in nonnutrient medium, a 3-hr refractory period ensues before pairing begins. A wave of cell division occurs concurrently with the onset of pairing. However, although all cells pair, the population does not double. This indicates that some cells do not divide and yet are capable of pairing. Apparently division per se is not required for pairing but does occur in most of the cells. Autoradiographic analysis demonstrates that the cells that divide before pairing were at a stage in the cell cycle beyond the initiation of macronuclear replication at the time they were transferred to nonnutrient medium. Cells that did not divide were in G1 at the time of shift-down. Thus, neither replication nor division is required to be able to fuse. However, since fusion occurs only in G1 and most cells are not in G1 at the time of shift-down, a traverse of the cell cycle is required. Shift-down induces G1 arrest and preparations for the mating reaction. Mixing the cells induces a synchronous wave of division for cells beyond the G 1 S interface. Preparations for the mating reaction occur independently of but simultaneous with the preparations for cell division.

A physicochemical analysis of conjugation in Tetrahymena pyriformis

Abstract The process of conjugation in Tetrahymena pyriformis is a useful model system for investigating mechanisms of cellular recognition, adhesion and fusion. As a first step in the biochemical analysis of this process, we have examined the effects of (a) nutrients; (b) metal ions; (c) several pharmacological agents (actinomycin D, cycloheximide, colchicine, theophylline, dithiothreitol and caffeine); and (d) temperature. We find that: 1. 1. While the complete nutrient medium inhibits conjugation, no single compound or group of compounds of the defined medium [1]produces any inhibition. 2. 2. At least trace amounts of Ca2+ are required. 3. 3. All of the pharmacological agents tested, except actinomycin D, inhibit both the preparations for conjugation and pair formation itself, indicating a requirement for both protein synthesis and low intracellular levels of cAMP, as well as the involvement of microtubules. 4. 4. While actinomycin D inhibits the preparations for conjugation, its addition after cells have begun to pair does not block further pairing. This result suggests that a stable RNA which is required for conjugation is produced during the preparations for conjugation. 5. 5. Paired cells may be disrupted for the first 1-1 1 2 i h after pairing by proteose peptone, cycloheximide, theophylline, and dithiothreitol. The cells undergo a transition 1 1 2 h after pairing which renders them resistant to these agents. 6. 6. The period of initiation (the time of starvation required to make cells competent to conjugate, the period of costimulation (the lag time preceding cell pairing after competent cells are mixed), and the rate of cell pairing are all temperature sensitive. Large changes in these parameters occur over narrow temperature ranges, possibly as a result of temperature-induced changes in membrane lipid composition or structure.

The conjugation junction of Tetrahymena: Its structure and development

The conjugation junction of Tetrahymena has been examined by thin sections, freeze fracture preparations, and by scanning electron microscopy. The junction is formed where the anterior tips of the pairing cells attach to one another. The structure is essentially a large disk composed of two face‐to‐face plasma membranes separated by a gap of extracellular space measuring about 50 nm. Rows of intramembrane particles are present at the boundary between the junction and ordinary cell cortex. These particles form a ring around the junction. Subjacent to each membrane is a thick mottled layer of material. Pores form in the junction at sites of membrane fusion. Though wider than long, these structures are actually bridges of cytoplasm that connect the conjugating cells. Pores fall into certain size and shape classes, indicating that membrane fusion is highly controlled in this system. At the level of the cytoplasmic bridge the submembrane material is compact and electron‐dense. Changes in the structure of the epiplasmic layer have been monitored as the normal cortex is modified during tip transformation and through formation of the mature conjugation junction. Evidence is provided that the submembrane layer plays a significant role in the regulation of pore formation. This cytoskeletal structure may also limit the extent of membrane fusion, thus controlling the size of the cytoplasmic channels.

G1 arrest and the division/conjugation decision in Tetrahymena.

Abstract The transformation from the asexual proliferative stage of Tetrahymena to the sexual stage, during which cells of complementary mating types pair and nuclear fertilization occurs, provides an opportunity to study the relationship between the division cycle and differentiation. Conjugation is induced in cells starved for at least 2 hr by mixing complementary mating types. To determine the effect of starvation on the cell cycle, dividing cells were selected from a log growth culture and stepped down to non-nutrient conditions. The G1 stage is operationally divisible into two sectors, A and B. In the A stage, cells arrest in nutrient-free medium. In the B stage, they proceed through the division cycle. Arrested G1A cells may conjugate directly when challenged with similar cells of a complementary mating type. It is thereby demonstrated that Tetrahymena cells in G1A can be directed to divide (nutrient conditions) or can be directed to differentiate (non-nutrient conditions plus complementary mating type) without an intervening division cycle. This rules out a requirement for reprogramming via chromosomal replication or cell division and suggests that G1A is a stage during which the division/differentiation decision is made in direct response to ambient conditions.

Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena

Abstract Tetrahymena cells of complementary mating types begin their pairing reaction 3 h after they are transferred to starvation buffer and mixed together. At the same time a division wave involving most but not all cells of both mating types is seen. If cells of each mating type are starved separately, no division wave occurs, thus indicating that cells of complementary mating types are capable of an interaction that leads to a reciprocal induction of a synchronous cell division. The basis for the interaction appears not to be mediated by a released factor but instead requires the physical presence of the differing cell types. Cells are sensitized to the interaction by a 2-h starvation period, at which time a surface transformation may occur, making possible a contact-induced stimulation. At 2 h after starvation all cells are either in G1 (arrested) or in G2, indicating that it is G2 cells that respond to the interaction. Nonetheless, G1 cells are capable of inducing the division on the complementary mating type.

Caspase-like Activity is Required for Programmed Nuclear Elimination during Conjugation in Tetrahymena

Abstract 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.

Differential density labeling and gradient centrifugation of Tetrahymena: A new procedure for selection synchrony☆

Abstract A simple technique has been developed which selectively separates late division and post-division cells from a culture of logarithmically growing, asynchronous Tetrahymena . The technique takes advantage of the division-related cessation of feeding. After a 5 min exposure to minute particles of tantalum, 90% of the population will have incorporated and concentrated the particles into food vacuoles. The remaining 10% constitutes a division population. Centrifugation through a Ficoll step-gradient separates the non-feeding and therefore less heavy division population from the feeding, interphase population. The separated cells show a peak of division at 170 ± 10 min and the cell number is doubled by the division. The bulk of DNA synthesis, as measured by 3 H-thymidine incorporation occurs between 30 and 120 min. At least with respect to the generation time and the duration of replication the selection procedure leaves the cell cycle unaltered.

A kinetic analysis of the memory of a developmental interaction: Mating interactions in tetrahymena pyriformis☆

Abstract The kinetics of an inductive intercellular interaction and the decay of the memory of that interaction during a developmental process have been analysed. The effects of three inhibitors (actinomycin D, cordycepin and α-amanitin) of RNA synthesis have been examined, in order to explore the role of RNA in both processes. In the protozoan Tetrahymena pyriformis, inductive intercellular interactions between starved cells of complementary mating types result in the ability to form heterotypic cell pairs. These interactions have been shown previously to be blocked by either actinomycin D or rapid agitation [1, 2]. We show here that the effects of both actinomycin D and agitation are restricted to the first phase of the inductive period. This implies that two separable processes take place during the inductive period: (1) a period during which cells are activated by the induction; (2) a maturation period during which preparations for pair formation in activated cells are completed. In contrast to the effects of actinomycin D, neither cordycepin nor α-amanitin blocks the induction of pair-forming ability. Since rapid agitation also independently blocks pair formation, the kinetics of the decay of the memory of the inductive interactions may be analysed by interrupting pairing for various lengths of time by agitation. In the absence of any inhibitors, the decay of the memory is a random, discrete event for any cell, with a half-time of 3.9 ± 1.4 h. While α-amanitin has only a moderate effect on the memory, both actinomycin D and cordycepin dramatically accelerate its decay, reducing the half-time to 35 ± 5 min. A biochemical model which accounts for these observations and which may be applicable to other developmental systems is proposed.