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Protistology 16 (2): 98-108 (2022) | doi:10.21685/1680-0826-2022-16-2-4 PPOtÎStOlOây
Original article
Changes of macronuclei structure during encystment in the ciliated protozoan Climacostomum virens
Bella P. Karajan1*, Olga G. Leonova2, Sergei O. Skarlato1 and Vladimir I. Popenko2
1 Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia
2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia
| Submitted February 18, 2022 | Accepted April 29, 2022 |
Summary
Ciliate cysts are an important object for investigating changes in cell morphology during cryptobiosis. In this work, we studied the ultrastructure of Climacostomum virens resting cysts using electron and confocal microscopy. The main attention was paid to the morphological changes in the macronucleus, nucleoli and chromatin. Vegetative cells, cysts 1-2 h after cyst wall formation, 2-months old cysts, one-year old cysts, and cells after excystment were investigated. The process of encystment in C. virens, as in other ciliates, is accompanied by a decrease in cell volume and the formation of a cyst wall. The volume of the macronucleus reduces, too. This reduction is likely to be related to the compaction of the macronuclear chromatin since extrusion of chromatin and nucleolar material into the cytoplasm during C. virens encystment was not observed. The nucleoli in young and 2-months old cysts retained their interphase morphology with well-defined fibrillar and granular zones. Only at the age of one year, the nucleoli in the cyst reduced in size and consisted of a dense homogeneous material. The chromatin structure in the cyst micronuclei was morphologically identical to macronuclear chromatin at most other cell cycle stages. Macronuclear chromatin in vegetative C. virens was mainly represented by individual bodies, 100—200 nm in size. As the age of the cysts increased, the chromatin bodies formed extended fibrils. In one-year old cysts, the entire space of the macronucleus was filled with fibrils 100—200 nm thick. Such fibrils resemble chromonemes in the chromosomes of higher eukaryotes and probably are formed by similar cellular mechanisms.
Key words: Climacostomum virens, resting cysts, ciliates, chromatin, nucleoli, confocal microscopy, electron microscopy
Introduction
Ciliate cysts are an important object for studying changes in cell morphology during cryptobiosis (Gutiérrez et al., 1990, 2001). The term "cryptobio-
https://doi.org/10.21685/1680-0826-2022-16-2-4
© 2022 The Author(s)
Protistology © 2022 Protozoological Society Affiliated with RAS
sis" was first used by David Keilin to describe the specific "state of an organism when it shows no visible signs of life and when its metabolic activity becomes hardly measurable, or comes reversibly to a standstill" (Keilin, 1959). Many organisms (both
Corresponding author: Bella P. Karajan. Institute of Cytology RAS, Tikhoretsky Ave. 4, St. Petersburg, 194064, Russia; bpkarajan@mail.ru
multicellular and unicellular) use this state to survive in unfavorable conditions. For example, it is known that resting cysts of soil ciliates and numerous other protists from different taxa can survive in permafrost for thousands of years at subzero temperatures (Shatilovich et al., 2015). Many complex biochemical reactions and molecular biology mechanisms are involved in the processes of encystment and excystment. For this reason, in the recent years the state-of-the-art methods such as transcriptomics, qRT-PCR and bioinformatic techniques were used to study the role of multiple signaling pathways (Pan et al., 2019; Shimada et al., 2021) and the microRNAome (Pan et al., 2020) in encystment. In particular, Pan et al. (2019) performed comparative transcriptomic analysis of dormant cysts and trophonts of Pseudourostyla cristata and found 2565 genes involved in the process. Among those, 1708 genes were upregulated and 857 were downregulated in the cyst (Pan et al., 2019). However, in general, our knowledge about the mechanisms of cryobiosis remains rather scanty.
Cell morphology changes drastically during encystment. Despite the fact that the investigated resting cysts show great morphological diversity, there are features common to all studied species. Most notable are the dramatic decrease in cell volume due to autophagy and dehydration of the cytoplasm, the metabolic inactivation, formation of the cyst wall, and structural rearrangement of nuclei, including the chromatin and the nucleoli (reviews: Gutiérrez et al., 1990, 2001; Verner and Rosati, 2011).
The unique feature of ciliated protozoa is the nuclear dualism. Each cell contains one or several germinal micronuclei that are transcriptionally inactive, and usually one large somatic polyploid macronucleus, which is transcriptionally active during vegetative growth. Unlike the nuclei ofhigher eukaryotes, the macronuclear genome is represented by a large number ofrelatively short DNA molecules (minichromosomes). All ciliated protozoa can be roughly divided into two groups according to the size of macronuclear DNA. The "gene-sized" species (e.g. Euplotes, Stylonychia, Oxytricha) typically have macronuclear DNA between 0.5 and 25 kb, whereas in "subchromosomal" species (e.g. Paramecium, Didinium nasutum, Bursaria trun-catella) macronuclear DNA molecules vary in size from several tens up to several hundred kb (reviews: Raikov, 1995; Jahn and Klobutcher, 2002). Each DNA molecule has two telomeric ends and an origin of replication.
It is now generally accepted that 3D architecture of the cell nucleus and the spatial organization of chromatin in it are the most important epigenetic factors that regulate genome expression. Thus, the study of ciliate cysts is of interest not only for understanding the processes occurring during cryptobiosis, but also for studying in vivo structural rearrangements ofthe genome, represented by many short DNAs, with a decrease in its activity.
In this work, we studied the ultrastructure of resting cysts of Climacostomum virens using electron and confocal microscopy. The main attention was paid to the morphology ofthe macronucleus, nucleoli and macronuclear chromatin. The morphology of macronuclei in early and late cysts was compared with the morphology of macronuclei of vegetative cells.
Material and methods
The laboratory strain of the Heterotrich ciliate Climacostomum virens was initially collected from a pond of the Valaam Island (Vaalam Archipelago, Ladoga Lake, Russia). Cells were cultured at room temperature in boiled tap water and fed 3 times a week with Tetrahymena pyriformis cultivated separately.
The resting cysts of Climacostomum virens
Encystment was induced by starvation ofcells for several days. After cessation offeeding, the cells were transferred to a refrigerator at 8 °C. After 24-48 h, 60-70% of the cells completely formed the cell wall of the cysts. Cysts with fully formed cyst wall were stored in culture medium at 8 °C. Vegetative cells, cysts 1-2 h after cyst wall formation, 2-months old cysts, one-year old cysts, and cells after excystment were collected for experiments.
Electron microscopy
Ciliates were fixed for 1 h on ice in the darkness with a 1:1 mixture of 4% glutaraldehyde in 0.1 M phosphate buffer and 2% osmium tetroxide in the same buffer. The mixture was prepared from stock solution immediately before use. Following fixation, the material was washed in 3 changes of the buffer and allowed to warm up to room temperature. The material was then pre-embedded in 2% agar gel, and agar blocks were dehydrated in a series of alcohol and acetone and embedded in Epon—Araldite mixture
y §
a
Fig. 1. Confocal images of Climacostomum virens in different states. a — Vegetative interphase cell; b — 2-month old resting cyst; c — one-year old cyst. d, e — macronuclei of interphase cell (d) and one-year old cyst (e) at higher magnification. Arrows point to micronuclei. Scale bars: a, b, c — 50 ^m; d, e — 5 ^m.
according to a standard procedure. The 50 to 70 nm thick sections were cut with an "Ultratome III" ("LKB"/Uppsala/Sweden) and mounted on electron-microscopic grids covered with a freshly ionized carbon-formvar supporting film. The sections were stained with aqueous uranyl-acetate and lead citrate solutions and studied in electron microscope ]EM-1OOCX ("]EOL"/Tokyo/]apan) at 80 kV accelerating voltage. For statistical analysis, 5 to 8 independent ultrathin sections of 3 different cells were measured. Areas were measured using the ImageJ software (https://imagej.nih.gov/ij/).
Confocal microscopy
The cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.3, washed 3 times in the same buffer and mounted in "Slowfade" medium ("ThermoFisher Scientific") containing 1 ^g/ml DAPI. Confocal images were acquired in a Leica DMI 6000B confocal microscope equipped with a Leica TCS SP5 laser scan unit ("Leica Microsystems", Mannheim, Germany). DAPI was excited at 405 nm and emission was detected in the 417—481 nm range.
Results
Confocal images of an interphase vegetative C. virens cell, 2-months old resting cyst and one-year old cyst are shown in Fig. 1. Each cell contains many (up to 14) micronuclei about 1 ^m in size and one large worm-like macronucleus. The resting cysts are surrounded by well-formed cyst walls. It is clearly seen that the volume of the resting cysts is smaller than the volume of the interphase vegetative cell (Fig. 1, a-c). The volume of the macronucleus also decreases, mainly due to shortening in length (more than 300 ^m in interphase macronuclei, less than 200 ^m in one-year old cysts), while the average diameter of the macronucleus does not change significantly. The mean diameters measured on confocal images were 14.5+1.0 ^m, 13.2+2.7 ^m and 10.6+1.1 ^m for interphase cells, 2-months old and one-year old cysts, respectively.
At higher magnification, changes in the structure of macronuclear chromatin are visible on confocal micrographs. Chromatin structures look like small granules in the interphase macronucleus, whereas in the one-year old cysts they have the appearance of thick threads (Fig. 1, d, e).
Fig. 2. Ultrathin sections of vegetative interphase C. virens cells. a — Fragment of a macronucleus; b — nucleolus; c — micronucleus. Abbreviations: n — nucleoli, f — fibrillar nucleolar component, g — granular nucleolar component. Scale bars: a, c — 1 ^m; b — 0.5 ^m.
Electron microscopic ultrathin sections show that macronuclear chromatin in vegetative interphase C. virens cells is organized into compact chromatin clumps 100—200 nm in size (Fig. 2, a). Some ofthem are located separately; others form short fibrils of 2-6 closely connected chromatin bodies. Macronuclei are surrounded by typical nuclear envelopes, consisting of inner and outer nuclear membranes with numerous porous complexes (Fig. 2, a). Nucleoli of 600-700 nm in diameter have a rounded shape. In the nucleoli, the fibrillar and the granular components are clearly distinguishable (Fig. 2, b). Micronuclei are filled with densely packed inactive chromatin (Fig. 2, c).
In macronuclei of young cysts fixed 3 h and 22 h after the formation ofthe cyst wall, the percentage of individual chromatin bodies decreases and the proportion of thick chromatin fibers increases (Fig. 3). The chromatin fibers composed of 7 and more chromatin bodies in 22-h old cysts are 126+26 ^m thick and are morphologically similar to chromonemes observed electron microscopically in chromosomes of higher eukaryotes (Zatsepina et al., 1983; Cook, 1995). Lipid granules appear in the cytoplasm of 22-h old cysts (Fig. 3, b).
In mature 2-month old cysts, the process of chromatin condensation continues (Fig. 4). Very few single chromatin bodies are present in macronuclei. The nucleoli become smaller in size, but fibrillar and granular components are visible in some nucleoli. Lipid granules are present in the cytoplasm and sometimes occupy fairly large areas ofthe cytoplasm (Fig. 4, b). The structure of micronuclei remains approximately the same as in the interphase cells and in 22-h cysts (Fig. 2, c; Fig. 3, c; Fig. 4, a).
Pronounced changes in the morphology of C. virens macronuclei were observed in cysts aged 3 months or more. An ultrathin section of a one-year old cyst with typical macronucleus morphology is shown in Figure 5. The nuclear membrane becomes folded, with poorly visible pores. Neither individual chromatin bodies nor short 100-200 nm chromatin fibers of 2-6 chromatin bodies were observed. The entire chromatin is represented by long chromonema-like fibers about 180 nm thick, forming an extensive network in the space of the macronucleus (Fig. 5, a). Nucleoli are represented by small rounded structures about 380 nm in size. The fibrillar and granular components are not distinguishable in them (Fig. 5, b). The dimensions
Fig. 3. Ultrathin sections of young resting C. virens cysts. a, b — Fragments of macronuclei in a 3-h old cyst (a) and a 22-h old cyst (b); c — micronucleus in the 22-h old cyst. Abbreviations: n — nucleoli, lg — lipid granules in the cytoplasm. Scale bars: 1 ^m.
of the nucleoli and the thickness of chromonema-like fibrils in interphase cells and resting cysts of C. virens, measured on ultrathin sections, are given in Table 1.
It should be noted that the morphological changes described above are reversible. The morphology of cellular structures is quickly restored after excysment. Fig. 6 shows an ultrathin section of a C. virens ciliate 24 h after excystment. The structure of both macronuclear chromatin and nucleoli is
virtually the same as in vegetative interphase cells (Fig. 2).
To describe quantitatively the structural changes of chromatin during encystment, we divided all chromatin structures observed on ultrathin sections into three groups and measured the area occupied by these structures. These three groups are: (1) individual chromatin bodies; (2) thick fibrils of 2-6 aggregated chromatin bodies; and (3) chromonema-like fibrils of 7 or more chromatin bodies. The
Fig. 4. Ultrathin sections of a 2-month old C. virens cyst. a — Micronucleus, b — macronucleus. Abbreviations: n — nucleoli, lg — lipid granules in the cytoplasm. Scale bar: 1 ^m.
relative area ofthese structures and their distribution in interphase macronucleus, as well as in the resting cysts of different age and after excystment are presented in Table 2 and in Fig. 7.
Discussion
The resting cysts studied so far have a great morphological diversity. At present, detailed morphological data on resting cysts of ciliates are available for about 40 species (Verni and Rosati, 2011), although ultrastructural studies of changes ofthe chromatin structure in the cyst macronucleus have been carried out for a smaller number of species.
The data obtained in our study show that the process of encystment in C. virens, as in other cilia-
tes, is accompanied by a decrease in cell volume and the formation of a cyst wall. The volume of the macronucleus reduces, too. In one-year old cysts, it decreases by about 2 times compared with the vegetative macronucleus. This decrease is likely to be related to the compaction of the macronuclear chromatin.
Extrusion of chromatin and nucleolar material into the cytoplasm during encystment has been observed in some species of ciliates (Beers, 1946; Martín-González et al., 1991; Frenkel, 1992; Bencat'ová et al., 1996; Popenko et al., 1998; Diaz et al., 2003; Akematsu and Matsuoka, 2008; Li et al., 2016; Gong et al., 2018). However, in C. virens cysts, we observed neither extrusion of chromatin nor nucleolar material. The nucleoli in young and 2-month old cysts retained their interphase morphology with well-defined fibrillar and granular
Table 1. The size of nucleoli and the thickness of chromonema-like fibrils in interphase cells and resting cysts of Climacostomum virens, measured on ultrathin sections.
Dimentions of nuclear structures Interphase vegetative cell 3-h old cyst 22-h old cyst 2-month old cyst One-year old cyst
The size of nucleoli (|jm) 546±169 455±124 388±96 335±66 379±70
Thickness of chromonema-like fibrils (|m) - - 126±26 121±22 183±37
Fig. 5. Ultrathin sections of a 1-year old C. virens cyst. a — Macronucleus, b — nucleolus. Abbreviations, n — nucleoli. Scale bars: a — 1 ^m, b — 0.5 ^m.
zones (Figs 2, 4). Only in one-year old cysts, nucleoli diminished in size and were composed of dense homogeneous material. It indicates that rRNA synthesis occurs in C. virens cysts for at least one to two months.
The micronuclei in resting cysts look almost the same as those in vegetative cells. The inactive chromatin structures in the cyst micronuclei are morphologically identical to micronuclear chromatin at most other cell cycle stages (Figs 2-4).
Condensation of macronuclear chromatin is an essential feature of the encystment process (Kawakami and Yagiu, 1963; Grimes, 1973; Walker and Maugel, 1980; Gutiérrez and Perez-Silva,
1983; Verni et al., 1984; Dallai et al., 1987; Martín-González et al., 1991; Frenkel, 1992; Popenko et al., 1998; Li et al. 2017). In most of the studied species, the structures of chromatin in the cyst macronuclei look like large amorphous compact chromatin masses or, as in stichotrichous ciliates, large spheroidal bodies. But macronuclear chromatin in resting C. virens cysts looks very different.
In vegetative C. virens macronuclei (Fig. 2), chromatin structures are mainly represented by individual bodies, 100—200 nm in size. As the age of the cysts increases, the chromatin bodies form more and more extended fibrils. In one-year old cysts, the entire space of the macronucleus is filled with
Table 2. Relative area (%) occupied by different chromatin structures on ultrathin sections of Climacostomum virens macronuclei.*
Stage Individual chromatin bodies Fibrils of 2-6 aggregated chromatin bodies Chromonema-like fibrils of 7 or more chromatin bodies
Interphase vegetative cell 45±5 55±5 0
2-month old cyst 6±1 51±6 43±7
1-year old cyst 0 0 100
Young cell 1 day after excystment 17±3 46±4 37±4
* Total area of all measured chromatin structures on ultrathin sections was taken for 100%
Fig. 6. Ultrathin section of a macronuclear fragment in the vegetative cell 24 h after excystment from a one-year old C. virens cyst. Abbreviations: n — nucleoli. Scale bar: 1 ^m.
fibrils 100-200 nm thick (Table 2). Such fibrils are morphologically similar to chromoneme fibrils in the chromosomes ofhigher eukaryotes (Zatsepina et al., 1983; Cook, 1995). Formation ofchromonema-like fibrils was also observed in the resting cysts of Bursaria truncatella and Didinium nasutum (Popen-ko et al., 1998; Karajan et al., 2003).
These results can be explained by the fact that the macronuclear genome of these ciliates, in contrast to, for example, hypotrichous species, is represented by DNA molecules of "subchromosomal" size of ~50-1700 kb. Chromatin bodies of100-200 nm in size are a typical form of chromatin organization for many "subchromosomal" ciliate species and can be considered as analogues of chromomeres or topologically organized domains (TADs) in the nuclei of higher eukaryotes (Leonova et al., 2021). It is known that chromatin bodies have a radialloop organization similar to the chromomeres of higher eukaryotes. During incubation in hypotonic solutions, they gradually decompact, forming a halo of chromatin nucleosomal fibrils around the central electron-dense part (Martinkina et al., 1983; Tikhonenko et al., 1984; Borkhsenius et al., 1988; Karajan et al., 1995; Popenko et al., 2015).
Complexes oftelomeric ends ofDNA molecules with telomere-binding protein TeBP (Murti and Prescott, 1999, 2002; Jönsson et al., 2002) play
the role of"organizing centers" around which chromatin bodies are formed. Loops of nucleosomal chromatin fibrils are formed around such "organizing centers", either by the principle ofloop formation in TAD (Razin and Gavrilov, 2018) or by attachment of DNA segments to proteins of the "organizing center" (Cook, 1995; Novikova and Popenko, 1998). The adhesive properties of the "organizing centers" and the nucleosomal fibrils around them are different. Therefore, with a decrease in activity during starvation or during encystment, the more adhesive organizing centers of chromatin bodies will stick together. Due to steric restrictions, further aggregation of chromatin bodies will occur with the formation ofcylindrical structures — "chromonema-like" fibrils, 100-200 nm thick (Novikova and Popenko, 1998; Leonova et al., 2021). The proposed model of chromatin organization in the macronuclei ofciliates of"subchromosomal" species (Leonova et al., 2021) is similar to the model of chromonema formation proposed by Cook (1995). The essential difference is that, in Cook's model, transcription factories are located in the central part of chromomeres (Cook, 1995) while quadruplexes oftelomeric DNA in complex with telomere-binding protein are located in the centers ofmacronucleus chromatin bodies (Leonova et al., 2021).
Fig. 7. Relative area occupied by different chromatin structures on ultrathin sections of Climacostomum virens macronuclei in vegetative cells, 2-month old cysts, one-year old cysts and vegetative cells 24 h after excystment. (Total area of all measured chromatin structures on ultrathin sections was taken for 100%).
Thus, it can be assumed that in "subchromoso-mal" species of ciliates, such as B. truncatella, Didi-nium nasutum, C. virens, the condensation of chromatin during a decrease in activity (during starvation) and encystment are based on the same cellular mechanisms.
Acknowledgements
This research was funded in part by the Budgetary Program # AAAA-A19-119020190091-5 at the Institute of Cytology RAS (B.P.K. and S.O.S.).
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