Jpn. J. Protozool. Vol. 38, No. 2. (2005) 153 Original How Paramecium cells die under a cover glass? Yoshiomi TAKAGI1,*, So KITSUNEZAKI2, Tae OHKIDO1,$ and Rie KOMORI1,# 1Department of Biological Sciences, Nara Women’s University, Nara 630-8506, Japan, 2Department of Physics, Nara Women’s University, Nara 630-8506, Japan SUMMARY were broken, and the cytoplasmic contents flew out. Until the last moment of the cell rupture, and The dying process of Paramecium tetraurelia sometimes even after that, ciliary beating was ob- cells under a cover glass without supporting pillars served somewhere locally on the cell membrane. was described. The initiation of the process was Among the cilia, those at the cytopharynx were the defined by the cessation of swimming, and the first to stop beating so that food vacuole (FV) for- termination by cell rupture. The process required 6 mation stopped somewhat earlier than the cellular min on average, ranging from 1 to 11 min. The first disorganization. Cessation of the contractility of symptom of death was the formation of a small contractile vacuoles (CVs) occurred a little bit later bleb, a local swelling of the outer cell membrane. on average than the cessation of FV formation. The number of blebs increased, each bleb grew, and Anterior and posterior CVs were not related to each neighboring blebs fused to form a larger bleb. By other in the timing of loss of contractility and in the supplying water, the blebs disappeared only when preceding pulsating cycles. Finally, we calculated their size and numbers were relatively small, in- the physical pressure to burst the cell at about 950 dicative of a commitment point to death. Finally atm and estimated the actual pressure to be at the outer membrane and then the inner membrane 470~2,000 atm. *Corresponding author Tel: 075-791-6846, E-mail: [email protected] Present addresses: *1-1 Kitadacho, Kamitakano, Sakyo-ku, Kyoto 606-0036 $2-277 Mozu-Honmachi, Sakai, Osaka 591-8036 #Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Shido, Kagawa 769-2193 Received: 20 June 2005; Accepted: 24 Aug 2005. 154 How Paramecium cells die under a cover glass? INTRODUCTION that of (b) is death by heat or pressure. It remains uncertain whether (a) and (b) are fundamentally People used to say that biology is the science different phenomena or not. It is natural, among of living organisms, not dead substances. Items living organisms, that a dead body will eventually destined for death or extinction such as the tail of a disappear (even cells killed by desiccation cannot tadpole during metamorphosis, the cuticle of an be a permanent preparation unless treated with insect pupa during molting, or the macronucleus of chemical fixative). For example, when we studied the ciliate during sexual process were not of inter- death by chemicals, we would observe many dead est until the concept of apoptosis (Kerr et al., cells some hours after the treatment, but no corpses 1972) became widely accepted. That caused a on the next day. It should be asked, therefore, how revolutionary change in biology by introducing cells die and how long the dead cells keep their death as an essential part of the living system. shape. However, there are still few investigations on the The length of time during which we can ob- dying process of protozoan cells, although the dy- serve dead cells varies according to the situation. ing process in apoptotic cells has been described But we have no answer to such questions as “Are abundantly, and the mechanism of genetically con- there any relationships between the cause of death trolled cellular death is now one of the most fasci- and the process of death, between the kind of nating biological themes. chemicals and the process of death, and between We found ourselves unable to answer the the concentration of chemicals and the persistence question of “how Paramecium cells die when they time of dead cells?”. are at an extremity of senescence”, although we Before answering such questions, we should had long engaged in examining aging and lifespan know the death process in detail. We here paid in Paramecium (Takagi and Yoshida, 1980; Ta- attention to the simple occasion of death by pres- kagi and Kanazawa, 1982; Takagi et al., 1987a, sure under a cover glass. We observed more than 1987b; Takagi, 1988, 1999; Komori et al., 2004, 100 samples of dying Paramecium cells under a 2005). We noticed that we had little knowledge cover glass without supporting pillars, depicted the about the dying process of Paramecium, not only sequence of the death process, and estimated the at senescence but also at more commonly occur- pressure required for cell rupture. ring accidental death from physical, chemical and biological causes, although we had frequently met death of Paramecium caused by competition for MATERIALS AND METHODS survival (Maruyama et at., 1996, 2001; Maruyama and Takagi, 1997), by UV (Yamamoto et al., 1997, The wild-type stock 51 of Paramecium tetra- 2005), and by toxins contained in a component of urelia was used. culture medium (Tokusumi and Takagi, 2000; Cells were cultured in 0.5% phosphate- Mizobuchi et al., 2003). buffered wheat grass powder (Pines Int., Inc., We have noticed so far that there are 2 forms Lawrence, KS) infusion supplemented with 0.5 of death: (a) death leaving the shape of the dead mg/l stigmasterol and inoculated with Klebsiella cell, and (b) death not leaving the shape of the pneumoniae 2 days before use (Mizobuchi et al., dead cell. A typical death form of (a) is death by 2003). A small sample of cells at the stationary desiccation, which is identical to the familiar proc- phase of growth were transferred to SMBIII ess of making permanent cell-preparations, and (Miyake, 1981), a saline solution, in wells of de- Jpn. J. Protozool. Vol. 38, No. 2. (2005) 155 pression slide for washing. broken, and the cytoplasmic contents flew out. A 10 µl drop of the saline solution including a definite number of less than 10 cells was put on a Dying process in detail clean slide glass, and a cover glass of (1.8 cm)2 in <Cell morphology> area and 0.1 g in weight was put on the drop with- After the cells stopped swimming, a small out pillars. A single cell was chased until it died bleb, an extrusion of the outer cell membrane was and the dying process was observed, paying spe- formed as the first symptom of the death process. cial attention to cell morphology, ciliary beating, The region where the first bleb was formed was food vacuole (FV) formation and contractile vacu- not fixed; every region on the cell surface ap- ole (CV) activity. We observed more than 100 peared to have an equal chance to form a bleb. dying cells in all. Video-recordings of cells were The number of blebs increased and small neigh- used for the observation of CVs. boring blebs fused to form a larger bleb. A typical Handling and observation were performed at morphological change is shown in Fig. 1. If water room temperature, which was usually in the range was supplied from a cover glass edge when blebs of 22~28ºC. were smaller in size and fewer in number, the blebs disappeared: the cell restored its normal shape and began to swim normally. The point of RESULTS no-return or the commitment point appeared to exist somewhere during the bleb-growing process. Sequence of death The cells at stages B and C in Fig. 1 would re- Paramecium cells which were swimming ac- cover, but thereafter would not. Cilia were regu- tively under a cover glass without pillars stopped larly located on the surface of the blebs, i.e., on the moving before long. Although the time to the outer membrane. Trichocyst-discharging was stoppage varied greatly among cells because of rarely observed during the dying process except at such factors as inconsistent vigor of each cell, vari- the moment of cell rupture. When observed, it was able rates of loss of water, contamination by debris outside the blebs. that supported the cover glass and so on, the period Concomitant with the enlargement of blebs, between the cessation of swimming and the cell enlargement of intracellular vacuoles occurred. rupture was 6 min on average, ranging from 1 to We were unable to identify each vacuole enlarged 11 min. except CVs which we will mention later. The first symptom of death was the formation <Ciliary beating > of a small bleb, a local swelling of the outer cell Paramecium cells stopped swimming, al- membrane. The number of blebs increased, each though cilia all over the cell surface continued to bleb grew, and neighboring blebs fused to form a beat. Sometimes cells that stopped forward or larger bleb. Cessation of ciliary beating at the backward swimming rotated at the resting position. cytopharynx (buccal cavity), which means the ces- This made it easy to observe the subsequent proc- sation of FV formation, tended to occur somewhat esses leading to death. Cilia on a part of the cell earlier than the cessation of the contractility of surface continued to beat until the moment of cell CVs. Ciliary beating was observed until the last rupture, and sometimes continued to beat after the moment of the cell rupture at least somewhere cytoplasm began to flow out (Fig. 1F). locally on the cell surface. Finally the outer cell <Food vacuole (FV) formation> membrane and then the inner cell membrane were Although FVs under formation were not al- 156 How Paramecium cells die under a cover glass? Fig.