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12УДК 57.086.3:582.282.23
ЦИТОЛОГИЧЕСКОЕ ИЗУЧЕНИЕ ДИНАМИКИ МИГРАЦИИ КЛЕТОЧНЫХ КОМПОНЕНТОВ В ХОДЕ ПОЧКОВАНИЯ CRYPTOCOCCUS NEOFORMANS
''Степанова A.A. (зав. лаб.)*, ''Васильева Н.В. (директор института, зав. кафедрой),2Ямагучи M. (главный специалист), 2Чибана Х. (профессор), 'Чилина Г.А. (зав. лаб.), 'Босак И.А. (с.н.с.)
1НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия; 2Центр исследований по медицинской микологии, Университет г. Чиба, Япония
©Коллектив авторов, 2018
Представлены данные по ультраструктурным аспектам динамики миграции компонентов клетки в ходе in vitro почкования дрожжевых клеток штамма (РКПГУ-1067) Cryptococcus neoformans. Материнские клетки С. neoformans содержат одно интерфазное ядро, небольшое число мелких митохондрий, плотный цитозоль, многочисленные свободные рибосомы, одну крупную вакуоль, 1-2 мелких ли-пидных включений. В ходе почкования размеры ядра и ядрышка увеличиваются наряду с возрастанием уровня хроматизации. Процесс почкования материнской клетки сопровождается увеличением числа митохондрий без формирования митохондриального ретикулума.
Ключевые слова: in vitro, миграция органелл, почкование, Cryptococcus neoformans, ультраструктура
CYTOLOGICAL INVESTIGATIONS OF CELL COMPONENTS MIGRATION DINAMICS DURING BUDDING IN THE YEAST CRYPTOCOCCUS NEOFORMANS
'Stepanova A.A. (head of the laboratory), 'Vasilyeva N.V. (director of the institute, head of the department), 2Yamaguchi M.M. (grand-fellow), 2Chibana H. (associated professor), Chilina G.A. (head of the laboratory), Bosak I.A. (senior scientific collaborator)
'Kashkin Research Institute of Medical Mycology, NorthWestern State Medical University named after I.I. Mechnikov, St-Petersburg, Russia; 2Medical Mycology Research Center, Chiba University, Chiba, Japan
©Collective of authors, 2018
The ultrastructural aspects of cell component migration dynamics during in vitro budding of Cryptococcus neoformans yeast cells strain (РКПГУ-1067) were presented. The mother cells of С. neoformans before budding had one interphase nucleus, dense cytosol, numerous free ribosomes, one large vacuole and 1-2 storage lipids. During budding, the sizes of nucleus and nucleolus were increased along with the increase of level of chromatization. The process of С. neoformans budding accompany with increasing in number of mitochondria without the formation of one giant
* Контактное лицо: Степанова Амалия Аркадьевна, e-mail: amaliya.stepanova@szgmu.ru
organelle.
Key words: budding, in vitro, migration of organelles, Cryptococcus neoformans, ultrastructure
INTRODUCTION
Cryptococcus neoformans (Sanfelice) Vuill. is basidiomycetous yeast, opportunistic pathogen. Previously on ultrastructural level we investigated the yeast cells morphogenesis of this species in vitro and in vivo on the example of clinical [1-7] and natural [8] strains of this species. Special researches have been devoted the senescent and desiccated in vitro growing yeast cells of C. neoformans [9] and patterns of interactions with murine lung macrophages [10]. Before on the example of two species from one Cryptococcus [C. albidus: 11, C. laurentii: 12] and different genus [Malassezia pachydermatis: 13, Rhodotorula minuta: 14] we revealed differences in the organellography during yeast cells budding and made the assumption that this data may be important for the taxonomical and phylogenetic investigations. Thus, we decided continue this investigations on the example of another yeast species from genus Cryptococcus - C. neoformans.
MATERIALS AND METHODS
We studied strain (PKOTY-1067, Russian collection of pathogenic fungi) of C. neoformans isolated from HIV-infected patient. The virulence of strain PKnTY-1067 was determined as medium with LD50 = 105-106 cells/mouse after intravenous injection [15].
For fixation we used the part of fungal colony after 72 hours of cultivation on solid Sabouraud's agar medium at 37 °C. For TEM samples were fixed in 3% glutaraldehyde buffer and post-foxed in 1% osmium tetroxide, then dehydrated and embedded in epoxy resin. The ultrathin sections were cut by Ultratome LKB V and stained with uranyl acetate and lead citrate. The sections were observed on the transmission electron microscope (TEM) JEM 100 SX (Jeol, Tokyo, Japan).
RESULTS AND DISCUSSION
Transmission electron microscopy. Mother cells
before budding. The central part of cell occupied with the interphase nucleus, one large and several small vacuoles. Large vacuole was similar in size with nucleus (Fig. 1 a). Nucleus and large vacuole occupied the main volume of mother cells. The nucleus localized near cell wall and opposite to vacuole, was spherical (2,2 (m) in form, contain moderate level of randomly distributed diffuse chromatin and near nucleolar envelope condensed one (Fig. 1 a). Nucleolus spherical, large (0,5 (m), composed with similar amount of granular and fibrillar components, localized in contacts with the block of the condensed chromatin (Fig. 1 a, b, arrow), which connected with nuclear envelope. Numerous ribosomes were localized on the outer nuclear membrane.
Vacuole localized opposite budding scar (Fig. 3) and contain the randomly distributed thin-fibrillar material. The mother cells on this and another stages of development were not different among themselves in vacuolar content structure, which significantly distinguished this species from previously investigated strain of the C. albidus [11] and draw together with C. laurentii [12], M. pachidermatis [13] and Rhodotorula minuta [14].
Mitochondria localized uniformly in the yeast cells (Fig. 1 a, 3 a). The number of these organelles varied from
2 to 4 on median cell section. They were spherical (0,5 (m) and ellipsoidal (0,5x0,6 (m) in form, contain matrix with moderate electron density and numerous light chaotically oriented cristae. Mitochondrial matrix was identical electron density with cytosol. The storage substances in form of 1 or 2 small lipid droplets (Fig. 1 a), which localized in peripheral part of cytosol. For comparison, in the mother cells of C. albidus [11], C. laurentii [12] and R. minuta [14], which growth in exponential phase (after 24 hour growth in YPD medium 30 °C), storage substances were absent. Contrary, for mother yeast cells of M. pachidermatis [13], which grows in similar condition, were typical presence the single large lipid inclusion situated opposite the budding scar. For the mature yeast cells C. neoformans with different virulence after 7 days of sowing on the Sabouraud's agar [3] were typical presence of different types of numerous storage substances.
Cytosol was moderate electron density, contain numerous free ribosomes. Single rare small (from 30 to 60 nm) light vesicles were revealed near cell wall. This situation was typical for mother cells C. laurentii [12]. But in mother cells of C. albidus [11], M. pachidermatis [13] and R. minuta [14] before budding, which growth in another condition as last species, the secretory vesicles with the same or another morphology in this and another stages were numerous.
Single cisterns of Golgi and microbodies absent on this in another developmental stage of budding. In the yeast cells C. neoformans after 7 days of beginning of infection in mouse lung and brain independent of its virulence [3], microbodies appear.
The cisterns of rough endoplasmic reticulum (ER) on all stages of budding were rare, short, straight or slightly curved, occupied peripheral position in cells (Fig. 1 c, 3). The small amount of rough ER were typical for mother cells of another basidiomycetous yeast species [C. albidus: 11; C. laurentii: 12, M. pachidermatis: 13; C. neoformans: 1-3, 8 and etc.].
Plasma membrane was in close contact with the electron-transparent thin (0,17 (m) light cell wall (Fig. 1 a) which supply with single budding scar (with mean diameter 0,6 (m). Cell walls whit median electron density, composed with 2 layers (inner thick dark with irregular outer surface and outer thin light).
Budding was started by development in the scar region evagination of plasma membrane and cell wall, which contain cytosol and free ribosomes (Fig. 1 d). In this period, the nucleus migrated from basal part of mother cell in its median part and localized near vacuole (Fig. 3). Soon during the isodiametrical growth the spherical bud was formed (Fig. 1 e, 3). The sizes of nucleus and nucleolus become larger. In nucleus the level of condensed chromatin increased significantly (Fig. 1 f, g), what was correlated with initiation of chromosome duplication (DNA synthesis) during budding. The analogous situation also took place with in vitro growing cells in species C. albidus [11]; C. laurentii [12], M. pachidermatis [13] and R. minuta [14]. In nucleolus the number of granular component was distinctly increased.
The numbers of mitochondria were increased slightly (before 5-6 on median section) (Fig. 1 f, g) without formation of mitochondrial reticulum which concentrated near nucleus, what was typical for budding cells of another yeast species [C. albidus: 11; C. laurentii: 12, R. minuta: 14].
The development of mitochondrial reticulum was typical for cells of strong virulent C. neoformans strains which infected mouse brain after seven days of experiments [3, 4]. Contrary, in the yeast cells of M. pachydermatis [14], the number of mitochondria, peculiarity of its topography and ultrastructure during budding were constant. In a whole, the activation of nucleolus and chondriome correlated with synthesis of cell wall and polysaccharide capsule, cytosol, free ribosomes and another cell component, which migrate in the developing bud.
During the C. neoformans bud growth, the cytosol, numerous free ribosomes, several single small mitochondria, small vacuoles (Fig. 1 d, e), rare short ER cisterns and rare single secretory vesicles were passes in its content from mother cell cytosol through isthmus. When the bud is about half size of the mother cells, the nucleus situated near isthmus (Fig. 1 e, f) and then migrated from it's in the developing bud content. In this period the isthmus was wide (1,2 (m), and large nucleolus (0,70 (m) was visible in the part of nucleus which localized in mother cell cytosol. According the data in literature, during of C. laurentii [10] yeast cells budding, the nucleus was revealed in the bud content and undergo mitosis in isthmus after which one nucleus stay in bud and another return in mother cell. According the data in literature similar pattern of nucleus behavior was typical for the budding cells C. neoformans [16].
After division completion, we observed one strongly chromatizated irregular in form and small sized (1,1x0,9 (m) nucleus in mother cell and another in bud cytosol (1,4x0,8, Fig. 2 a). Later the size of nucleolus in bud and mother cell gradually increased and their forms became spherical.
The mother and daughter cells during and after separation. After mitosis, cytokinesis in middle part of isthmus was start. In this region the wedge-shaped electron transparent septum was simultaneously formed during the centripetal grow before central part (Fig. 1 h, arrows) At last, in the middle part of isthmus thick (0,2), continuous and light septum was formed (Fig. 1 I arrow, 2 a). The subsequent daughter cell separation from mother one passed through the middle layer of septa (Fig. 1 j arrow, 2 b, c). In the mother cells topography and number of organelles after this process returned in condition before budding (Fig. 3). One spherical (1,6 (m) nucleus with higher level of chromatization localized in central part of daughter cell (Fig. 2 c, e). It contains one nucleolus (Fig. 2 e) in which granular component predominate. Several small spherical vacuoles (Fig. 2 d-e) contain the thin-fibrillar material and membranous fragments. The number of spherical (0,5-0,6 (m) mitochondria (Fig. 2 e, g) varied from 2 to 3. The number of free ribosomes and cisterns of ER was similar with the mother cells after budding. The moderate number (from 15 to 18) of light secretory vesicles present on the median cell section (Fig. 2 d)). They localized in the cytosol of middle part of daughter cell. Single small lipid inclusions (Fig. 2 f, g) present in cytosol in this period. Soon separated daughter cell undergo by isodiametrical growth (Fig. 2 g), during which nucleus localized near cell wall (Fig. 2 g), large vacuole by the way of fusion of several small one was formed. In this time synthesis of the cell wall, cytosol, free ribosomes and proliferation of mitochondria take place. Simultaneously, the nucleus size was increase and level of chromatization decrease. Later, the nucleus and
Fig. 1. Ultrastructure of in vitro growing C. neoformans budding yeast cells. Explanation for this and another figures: B - bud, BC -budding cells; CW - cell wall, DC - daughter cell, ER - endoplasmic reticulum, Im - isthmus, M - mitochondrium(ia), MC - mother cell, MR - mitochondrial reticulum, N - nucleus, Nu - nucleolus, S - scar, St - septa, V - vacuole, Vs - vesicles.
vacuole localized near cell wall in basal part of cell. Thus, the transformation of daughter cell in mother was finished (Fig. 3).
Fig. 3. Schematic drawing of patterns of in vitro growing budding yeast cells of investigated yeast species.
CONCLUSION
Comparison of mother cell ultrastructure in cultures of three species from genus Cryptococcus - C. neoformans [data of the present work], C. albidus [11], C. laurentii [12] and R. minuta [14] demonstrated the common peculiarity in internal topography before (nucleus in basal part and vacuole opposite the scar) and during (migration of the nucleus from distal to lateral and opposite the scar and, contrary, transition of nuclei from apical part in distal one) budding (Fig. 3). Another topographical peculiarity (central-lateral) was typical for interphase nucleus in mother cell of M. pachydermatis [9]. For mother cells of all compared yeast species were typical the high level of vacuolization, moderate number of mitochondria and cistern of rough ER, abundance of free ribosomes, absence of single cisterns of Golgi and microbodies. Storage substances in budding cells of C. albidus [11], C. laurentii [12] and R. minuta [14] were absent. During yeast cells budding of M. pachydermatis [9] we revealed presence of one large storage lipid inclusion (Fig. 3), which localized
opposite scar (like vacuole in another investigated species). Several small lipid inclusions were present in budding yeast cells of C. neoformans [data of the present work], what may be caused by more long (72 hour) cultivation on another medium (solid Sabouraud's agar). Specific feature of yeast cells budding of C. albidus [11], C. laurentii [12] and R. minuta [14] were the development in mother cells and after separation in daughter cell (only in R. minuta) the mitochondrial reticulum (one giant mitochondrion) which presence deducible this process on energetically more high level. It was interesting that in C. neoformans [1, 4, 6] mitochondrial reticulum formed in tissular mature strong virulent yeast cells. In the mature yeast cells of C. neoformans [8] strains which isolated from natural condition giant mitochondrion absent. In this regard, it was actually to carry out to study the process of budding on the example strong virulent strain of this last species for the aim «put the point» in the question about correlation between level of the virulence on presence of mitochondrial reticulum.
In all investigated species cistern of rough endoplasmic reticulum in all stages of budding present in minor quantity, single cisterns of Golgi were absent. Moderate number of secretory vesicles we observed in budding yeast cells of all investigated species for the exclusion R. minuta [14]. In last species aggregations of numerous secretory vesicles present in all stages of yeast cells budding.
According our opinion, isolation in mother cell the nucleus from scar with the «using» of vacuole in C. neoformans [data of the present work], C. albidus [11], C. laurentii [12], R. minuta [14] and large lipid inclusion in M. pachydermatis [9] block the budding process and subsequent changes in topography between nucleus, vacuole and storage lipid (M. pachydermatis) was the first signal for starting of this process. For interphase nucleus of all investigated yeast species, were typical lower level of chromatization, which was typical for fungi [5, 6, 11, 12 and etc.] and correlated with lower number of chromosome. In all investigated species transition of mother cells to bud formation were accompanied by increasing of nucleus and nucleolus sizes and level of chromatization. In our opinion, nucleus activation correlated with the starting of mitosis and necessity activation of synthetic processes (synthesis of cytosol and free ribosomes) and organelles proliferation in the mother and daughter cells. For all investigated species, for exclusion M. pachydermatis, were typical specific activation of internal structure (proliferation of mitochondria, changes in its topography and etc.)
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Поступила в редакцию журнала 25.05.2017
Рецензент: Л.В. Филиппова
