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10УДК 57.086.3:57.012.4:582.282.23:591.165.2
УЛЬТРАСТРУКТУРНЫЕ АСПЕКТЫ МИГРАЦИИ КЛЕТОЧНЫХ КОМПОНЕНТОВ В ХОДЕ ПОЧКОВАНИЯ CRYPTOCOCCUS LAURENTII
''Степанова A.A. (зав. лаб.)*, 2Ямагучи М. (адъюнкт-профессор), 2Чабана Х. (ассистент профессора), 'Васильева Н.В. (директор института, зав. кафедрой)
1 НИИ 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, 2016
В статье представлены цитологические данные о миграции клеточных компонентов в ходе почкования дрожжевых клеток Cryptococcus laurentii в экспоненциальной стадии роста in vitro. В материнской клетке выявлены: интерфазное ядро с низким уровнем конденсированного хроматина, крупная вакуоль, плотный цитозоль, многочисленные свободные рибосомы, умеренное количество митохондрий, цистерн гранулярного эндоплазматического ретикулума (ЭР) и секреторных пузырьков. На этой стадии запасные вещества, одиночные цистерны Гольджи и микротельца отсутствовали. В ходе почкования материнской клетки мы обнаружили увеличение размеров ядра и возрастание степени его хроматизации, а также размеров ядрышка, количества митохондрий с формированием гигантской органеллы. С началом почкования ядро материнской клетки мигрировало из базальной части в латеральную, а затем в апикальную часть, где оно располагалось напротив рубчика. Вначале в области рубчика возникал вырост цилиндрической формы, который претерпевал изодиаметрический рост с формированием сферической почки. До митоза цитозоль, свободные рибосомы, небольших размеров митохондрии, мелкие вакуоли, короткие цистерны ЭР и несколько секреторных пузырьков мигрировали из материнской клетки в растущую почку. После перемещения ядра из материнской клетки в содержимое почки и последующего его деления, в области перешейка формировалась септа, по которой происходило отделение материнской клетки от дочерней.
Ключевые слова: замораживание-замещение, in vitro, Cryptococcus laurentii, почкование, ультраструктура, электронная микроскопия
ULTRASTRUCTRAL ASPECTS OF CELL COMPONENTS MIGRATION DURING BUDDING IN THE YEAST CRYPTOCOCCUS LAURENTII
'Stepanova A.A. (head of the laboratory), 2Yamaguchi М. (grand-fellow), 2chibana H. (assistant professor), 'Vasilyeva N.V. (director of the institute, head of the chair)
iKashkin Research Institute of Medical Mycology, Northwestern State Medical University named after I.I. Mechnikov, St. Petersburg, Russia; 2Medical Mycology Research Center, Chiba University, Japan
© Collective of authors, 2016
Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812)303-51-40
In this article the cytological data of the cell components migration during budding of the Cryptococcus laurentii yeast cells in exponential stage of in vitro growth were presents. In the mother cells we revealed the interphase nucleus with lower level of condensed chromatin, large vacuole, dense cytosol, numerous free ribosomes, moderate amount of mitochondria, cisterns of granular endoplasmic reticulum (ER) and secretory vesicles. In this stage, the storage substances, single Golgi cisterns and microbodies were absent. During budding of the mother cells we revealed the increasing of the sizes of nucleus with increasing the level of its chromatization and nucleolus sizes, the number of mitochondria with formation of giant organelle. At the beginning of budding, the nucleus in mother cell migrated from basal part to lateral and finally in apical area and localized near opposite scar. At first, in scar region the cylindrical protrusion was formed, which then undergo the isodiametrical growth with formation of spherical bud. Before mitosis, cytosol, free ribosomes, small mitochondria and vacuoles, short cistern of ER, several secretory vesicles migrated from mother cell to growing bud. After nucleus transition from mother cell in bud content and its mitotic division, the septum was formed in region of isthmus along which the separation of mother cell from daughter occur.
Key words: budding, Cryptococcus laurentii, electron microscopy, freeze-substitution, in vitro, ultrastructure
INTRODUCTION
Cryptococcus laurentii (Kufferath) Skinner - is basidiomycetous yeast, which was distributed in soil, air, seawater and contaminated wine, wheat, maise, grape, deep-frozen beans, phyllosphere of grasses, leaves of tropical plants and etc. Also C. laurentii reported from droppings and cloacae of pigeons. It was isolated as the etiologic agent of human skin infection, keratitis, endophthalmitis, lung abscess, chronical pneumonia, peritonitis, meningitis and catheter-associated fungemia [1-4; Banerjee P., et al. // Case Report. - 2013. - Vol. 31, Is.1, etc.]. C. laurentii was isolated from the blood of a patient given the diagnosis of ganglio-neuroblastoma [Averbuch D., et al. Med. Mycol. - 2002. - Vol. 40, №5]. This species was revealed in the dog with panninculitis and osteomyelitis, in the stomach of aborted equine fetuses. The purpose of this study was, first, on the example in vitro growing yeast cells of C. laurentii provide the ultrastructural investigations of organelle migration during the cardinal stage of morphogenesis - budding and, second, compare this data with the same obtained before for another species from genus Cryptococcus - C. neoformans [Kopecka M., et al. //Scripta Medica (Brno). -2000. - Vol. 73, №6], C. albidus [5] and another genus of basidiomycetous yeast - Malasseziapachydermatis [6].
MATERIALS AND METHODS
We investigated cultures of two C. laurentii strains (PKnrY-1014/B-292 from Russian collection of pathogenic fungi and IFM 50262 from Culture Collection of the Research Center of Pathogenic Fungi, Chiba University, Japan). The first strain was cultivated 10 days on solid wort agar at 28° C. For scanning electron microscopy, the part of fungal colonies with nutrient medium was fixed in 3% glu-taraldehyde (on 0,1 M cacodylate buffer) for 3 hour, post-fixed overnight in 1% osmium tetroxide in same buffer, dehydrated by ethanol series, critical-point dried (HCP-2) for 15 min, coated with gold and observed in JSM 35 (JEOL, Tokyo, Japan). The second strain was cultivated for 24 hour in YPD medium (1% (w/v) yeast extract, 2% (w/v) bactopepton and 2% (w/v) glucose on shaker at 30° C. For investigation of the living cells of cultures, we made the temporary preparation and investigation under the phase-contrast microscope (Olympus BH-2RFCA). For transmission electron microscopy (TEM), the cells were collected by centrifugation and sandwiched between two copper grids. Samples were then freeze-substituted in 2% osmium
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tetroxide/acetone at - 80° C for 48 hour and embedded in epoxy resin according to the method described before [7]. Ultrathin sections (70 nm thick) were cut with diamond knife then stained (uranyl acetate and lead citrate) and covered with Super Support Films (Nisshin EM, Tokyo, Japan) then observed with JEM-1400EX transmission electron microscope (JEOL, Tokyo, Japan).
RESULTS AND DISCUSSION
Phase-contrast microscopy. After 24 hour of growth on YPD medium, yeast cells were spherical or ellipsoidal (2-5 x 3-7 ^m), single or in small groups (Fig. 1 a) with monopolar budding. Hyphae and pseudohyphae were absent [1].
Scanning electron microscopy. Under the lower magnifications, the yeast cells in culture form the specific massive variable in size spherical and irregular yeast cells aggregations (Fig. 1 b, c, arrows). The developing yeast cells covered with polysaccharide capsule for which typical the slightly irregular surfaces textures (Fig. 1 d, arrows).
Transmission electron microscopy. Mother cells before budding. In central part of cell, we revealed the interphase nucleus and large vacuole, which were similar in size and occupied the main its volume (Fig. 1 e, 3 a). The nucleus localized in distal part of cell between vacuole and cell wall, was spherical (2,0 ^m) in form, contained moderate level of randomly distributed condensed chromatin. Nucleolus also spherical, large (0,5 ^m), composed with similar amount of granular and fibrillar components (Fig. 1 f). On the outer nuclear membrane the moderate number of ribosomes were revealed.
Large vacuole localized opposite budding scar and was characterized with the presence of specific tightly localized thin-fibrillar material (Fig. 1 e). The cells on this and another stage of development were not different among themselves in vacuolar content structure, which significantly distinguished this species from previously investigated strain of the C. albidus [5].
Mitochondria were in the periphery of cells near cell wall (Fig. 1 e). The number of organelles varied from 4 to 6. They were spherical (0,6 ^m) and ellipsoidal (0,5 x 0,6 ^m) in form and had moderate electron density matrix which was identical electron density with cytosol.
The storage substances in this and another stage of development were absent. This peculiarity was also typical for budding yeast of C. albidus [5], which cultivated for 24 hour in same conditions. It was interesting, that for yeast cells of M. pachidermatis [6], which grows in similar condition, were typical presence the single large lipid inclusion situated opposite the budding scar. The presence of large amount of different storage substances were typical for cells of C. neoformans with different virulence after 7 days of its sowing on the Saburaud's agar [8].
Cytosol with moderate electron density was rich with free ribosomes. Small number (from 3 to 6 on median cell section) single or in small groups (Fig. 1 f) small (60 - 70 nm) secretory vesicles with thin-fibrillar content were distributed in cytosol. Single cisterns of Golgi and microbodies were not found on all developmental stages. The cisterns of granular ER on this and subsequent stages of budding were rare, short, straight or slightly curved, localized near cell wall (Fig. 1 f). The small amount of granular ER were typical for mother cells of another basidiomycetous yeast species [C. albidus: 5; M.
pachydermatis: 6; C. neoformans: 8, 9]. For comparison, microbodies appeared in the yeast cells C. neoformans after 7 days of beginning of infection in mouse lung and brain independent of its virulence [8].
Plasma membrane was in close contact with the electron-transparent thin (0,17 ^m) light cell wall (Fig. 1 e, f) which supply with single budding scar (with mean diameter 0,6 ^m).
Budding. The process of budding was started by development in the scar region evagination of plasma membrane and cell wall, which contain cytosol and free ribosomes (Fig. 1 g, arrow). Later in this places was formed a cylindrical protrusion (Fig. 1 h, i, arrow, 3 b). For comparison, the budding of yeast cells in C. neoformans [Kopecka M., et al. // Scripta Medica (Brno). - 2000. - Vol. 73, №6; Yamaguchi M., et al. // J. of Electron Microscopy. - 2002. - Vol. 21, №1] also start with formation of similar protrusion. But we did not revealed its in budding yeast cells of C. albidus [5] and M. pachydermatis [6]. At this time, the nucleus migrated from basal part of mother cell in its median part (Fig. 1 h) and localized near vacuole. In this period several small vacuoles were appear in cytosol. Soon during isodiametrical growth of this cylindrical protrusion, the spherical bud was formed (Fig. 1 g, k, 3 c). In mother cell, the numbers of mitochondria were increased before 10 - 17 on median section (Fig. 1 j). They formed around nucleus and small vacuole distinctive «sheath» composed from closely contacted organelles (Fig. 2 a, 3 c). Perhaps, increasing the number of mitochondria and peculiarity of its topography in this early stage of budding was the evidence of development of giant organelle so called «mitochondrial reticulum». Formation of giant organelle around nucleus was revealed for budding cells C. albidus [5] and for strong virulent strains of C. neoformans growing in vivo (mouse brain after seven days of experiments [9]. Contrary, in the yeast cells of M. pachydermatis [6], the number of mitochondria, peculiarity of its topography and ultrastructure during budding were constant.
During subsequent stages of bud formation, the sizes of nucleus and level of condensed chromatin were increased (Fig. 2 c). Also the activation of nucleolus was revealed (increasing its size and volume of granular components). We obtained similar data for in vitro growing budding yeast cells of C. albidus [5] and M. pachydermatis [6]. The activation of nucleolus and chondriome correlated with synthesis of cell wall and polysaccharide capsule in developing bud and also cytosol, free ribosomes and another cell components, which migrate in the developing bud.
According the lower amount of secretory vesicles concentration in cytosol, the budding yeast cells of C. laurentii possible compared with similar of C. albidus [5] and C. neoformans [8, 9]. For comparison, in M. pachydermatis [6] these components of endomembrane system were not revealed.
During the bud growth, the cytosol, numerous free ribosomes, several single small mitochondria, small vacuoles (Fig. 2 b, c, d, 3 d), rare short ER cisterns and several secretory vesicles were pass in its content from mother cell cytosol. Directly in the isthmus cytosol, near nuclear outer membrane and vacuolar tonoplast possible revealed the single short microtubules (Fig. 2 d). This cytoskeleton components were also revealed in actively in vitro growing cells of C. albidus [5], M. pachydermatis [6]
Fig. 1. Phase-contrast (a), scanning (b - d) and transmission electron microscopy (e - k) of in vitro growing C. laurentii cells. Explanation for this and another figures: B - bud, CP - cylindrical protusion; DC - daughter cell, CW - cell wall, ER - endoplasmic reticulum; M - mitochondrium(ia), MC - mother cell, Mt - microtubule, N - nucleus, Nu - nucleolus, S - septum; Sc - scar; V -vacuole, Vs - vesicles. Scale: a - 6 |m, b - 500 |m, c - 100 |m, d - 3 |m, e - 2 |m, g, k - 0,5 |m, i - 0,4 |m, f, h, j - 1 |m.
Fig. 2. Ultrastructure of in vitro growing C. laurentii cells during (a - g) and after budding (h - k). Scale: a, b, c, e, f, I, k - 2 |m, d, g, h - 1 |m, j - 0,2 |m.
and C. neoformans [9; Yamaguchi M., et al. // J. of Electron Microscopy. - 2002. - Vol. 21, №1]. Previously, the detailed investigations of cell cytoskeleton system (microtubules and F-actin) during budding yeast cells of C. laurentii was carried out by Marek D., Gabriel M. and Kopecka M. [10]. These authors demonstrated, that cytoskeleton components play important role during budding of yeast cells of this fungal species and undergo the regular changes during this cardinal time of morphogenesis and are similar to those in C. neoformans.
When the bud is about half size of the mother cells, the nucleus situated near isthmus and then directly passes from its (Fig. 2 e, 3 e). At this time, the isthmus was more wide (1,25 ^m), and large nucleolus (0,75 ^m) was visible in the part of nucleus which localized in mother cell cytosol. According the data in literature [10], during of C. laurentii 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. Similar pattern of nucleus behavior was typical for the budding cells of another species of pathogenic fungus - C. neoformans [11].
After division completion, we observed one strongly chromatizated irregular in form and small sized (1,0 x 0,8 ^m) nucleus in mother cell and another in bud cytosol (1,4 x 0,8, fig. 2 f, 3 f). Later the size of nucleolus in bud (Fig. 2 g) and mother cell gradually increased and their forms became spherical.
The mother and daughter cell during and after separation. Soon after mitosis and during cytokinesis in middle part of isthmus, thick (0,17) light septum was formed (Fig. 2, h, j, 3 g). Cell separation passed through middle layer of septa. After this process (Fig. 2 i, 3 h), the mother cells ultrastructure, topography and number of organelles returned in condition before budding.
In central part of daughter cell revealed was spherical nucleus (1,5 ^m), one light median in size spherical vacuole (Fig. 2 i) with and thin-fibrillar material in its content. The number of spherical (0,6 ^m) mitochondria varied from 2 to 3. The number of free ribosomes, cisterns of ER and secretory vesicles was similar with the mother cells after budding. After separation from mother cell, the daughter cell undergo isodiametrical growth (Fig. 2 k), which was accompanied with formation in its apical part the central vacuole which localized apposite scar. In this stage synthesis of the cell wall, cytosol, free ribosomes and proliferation of mitochondria take a place. Simultaneously, the nucleus size was increase and level of chromatization decrease. Finally, they localized near cell wall in basal part of cell.
Fig. 3. Diagram showing the organelles migration during C. laurentii budding. a - mother cell before budding, b - g -mother cell during budding, h - mother and daughter cell cells after separation.
RESUME
Ultrastructural comparison of mother cell in cultures of two species from genus Cryptococcus - C. laurentii and C. albidus [5] in exponential phase of growth 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. Another topography was typical for interphase nucleus in mother cell of M. pachydermatis [6] - central-lateral. For mother cells of all compared four 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. Only for mother and budding cells of M. pachydermatis was specific the presence of storage lipid inclusion, which localized opposite scar (as vacuole in another species). According our opinion, isolation in mother cell the nucleus from scar with the «using» of vacuole in C. albidus [5], C. laurentii and large lipid inclusion in M. pachydermatis [6] block the budding process and subsequent changes in interrelations between nucleus^ vacuole and storage lipid 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 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, first, with grounding to mitosis and, second, with necessity activation of synthetic processes and organelles proliferation.
Budding in all species were accompanied by the synthesis of cytosol and free ribosomes. For all investigated species, for exclusion M. pachydermatis, were typical specific proliferation of mitochondria, changes in its topography, including formation of a «sheath» around
the nuclear envelope, which we associate with formation of one giant organelle. For comparison, during mother cell development of natural [13] and clinical [9] isolates of C. neoformans giant mitochondria were not developed. Thus, in general, in investigated pathogenic yeast species giant mitochondria developed during budding, but the M. pachydermatis [6] distinctly differ from this species by absence of chondriome proliferation in mother cells, which undergo the same process.
In mature and budding mother cell of all investigated yeast species, for exclusion of R. minuta [7], the number of secretory vesicles was small. As a rule, they not revealed near cells wall in developing bud and separated septa between the mother and daughter cell. Thus, in the yeast cells of this species its participation in cell wall synthesis and construction was questionable. Also not clear the source of secretory vesicles, so that the single cisterns Golgi we not revealed. Our data support the opinion of Vasilyev A. E. [Vasilyev A.E. // Bot. Journal - 1985. -Vol. 70, №9] that fungal cell was «mesocaryotic» and we often find the presence of prokaryotic and eucaryotic pattern of cell functioning. It was obvious, that in our objects all morphogenetic process may pass as in bacteria only with participation of plasma membrane, cytosol and free ribosomes. For conformation: the yeast cell budding in M. pachydermatis [6] pass only on the base of nucleus activation and without chondriome proliferation. Revealed abundance of secretory vesicles in budding yeast cells of R. minuta [7] may be demonstrated its differences from another investigated species in rate and quality of synthesis
and secretion of ferments and secondary metabolites.
Unfortunately, but we not observed directly process of division, but according the data another authors in C. neoformans [11, 12], M. pachydermatis [14] and R. glutinis [McCully E.K., Robinow C.F. // J. Cell Sci. - 1972. - Vol. 11] it was took places during nucleus moving through isthmus. During budding the yeast cells in C. albidus [5], nucleolus separate on equal parts during in isthmus region during nucleus migration. Previously we [5] mentioned, that the behavior and the fate of nucleolus during budding of yeast cells constant for member of one genus and, perhaps, may indicate the level of its evolutional advances and relations. In all four investigated yeast species after mitosis, one nucleus revealed in mother and another in bud. It was important, that exactly after mitosis the nucleus in mother and daughter cell posses with one nucleolus.
Our data demonstrated that, first, we revealed the similar pattern of changes in mother cells budding on example of two species from one genus Cryptococcus and, second, differences between this species and members of another genus of basidiomycetous yeast - Malassezia. It was obvious, that the data relative to the pattern of changes during budding of mother cells was very important and perspective for taxonomical investigations and actually continue its. In contrast, the regularity of organelles transition during budding in system mother cell — bud and ultrastructure of developing bud was identical for all investigates yeast species in depending of its taxonomical position.
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Поступила в редакцию журнала 15.06.2016
Рецензент: Богомолова Т.С. Ja
