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ЭЛЕКТРОННО-МИКРОСКОПИЧЕСКОЕ ИЗУЧЕНИЕ ДИНАМИКИ КЛЕТОЧНЫХ КОМПОНЕНТОВ В ХОДЕ ПОЧКОВАНИЯ RHODOTORULA MINUTA
''Степанова A.A. (зав. лаб.)*, 2Ямагучи M. (главный специалист), 2Чибана Х. (профессор), '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; 2 Центр исследований по медицинской микологии, Университет г. Чиба, Япония
©Коллектив авторов, 2017
Впервые представлены данные по ультраструктуре динамики компонентов клетки в ходе почкования дрожжевых клеток Rhodotorula minuta в экспоненциальной фазе in vitro. Материнские клетки R. minuta имеют одно интерфазное ядро, плотный цитозоль, многочисленные свободные рибосомы, высокий уровень вакуолизации, обильные секреторные пузырьки, однако цистерны Гольджи, микротельца и запасные вещества были выявлены в небольшом числе. В ходе почкования размеры ядра и ядрышка увеличиваются параллельно с возрастанием уровня хроматизации. Для процесса почкования R. minuta типично увеличение числа митохондрий с формированием одной гигантской органеллы. Плотная ассоциация рибосом с наружной мембраной митохондрий дрожжевой клетки свидетельствует о том, что митохондриальная активность возрастает в ходе ее почкования.
Ключевые слова: замораживание-замещение, in vitro, почкование, Rhodotorula minuta, ультраструктура
ELECTRON-MICROSCOPIC INVESTIGATION OF CELL COMPONENT DYNAMICS DURING BUDDING IN THE YEAST RHODOTORULA MINUTA
'Stepanova A.A. (head of the laboratory), 2yamaguchi M.M. (grand-fellow), 2chibana H. (associated professor), 'Vasilyeva N.V. (director of the institute, head of the chair)
'North-Western State Medical University named after I.I. Mechnikov: Kashkin Research Institute of Medical Mycology, Saint Petersburg, Russia; 2Medical Mycology Research Center, Chiba University, Chiba, Japan
©Collective of authors, 2017
The ultrastructure of cell component dynamics during budding of Rhodotorula minuta yeast cells at the exponential phase in vitro was presented for the first time. The mother cells of R. minuta had one interphase nucleus, dense cytosol, numerous free ribosomes, higher level of vacuolization, abundant secretory vesicles, but few Golgi cisterns, microbodies, and storage substances were revealed. During budding, the sizes of nucleus and nucleolus were increased along with the increase of level of chromatization. The increase in number of mitochondria with the formation of one giant organelle was typical for the process of R. minuta budding. Tight
* Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
association of ribosomes with mitochondrial outer membranes may suggest that mitochondrial activity would increase during its budding.
Key words: budding, freeze-substitution, in vitro, Rhodotorula minuta, ultrastructure
INTRODUCTION
Rhodotorula minuta (Saito) F.C. Harrison - is widely distributed basidiomycetous yeast, opportunistic pathogen. This species causes endophthalmitis [Gregory J.K., Haller J.A. Arch. Ophthalmol. 1992; 110], onychomycosis [1], systemic infection in a leukemic patient [Rusthoven J.J., et al. J. Infect. 1984; 8] and infection in hip joint prosthesis [Cutrona A.F., et al. Am. J. Orthop. 2002; 31]. It was also isolated from the central venous catheter of the AIDS patient [Goldani L.Z., et al. J. Med. Vet. Mycol. 1995; 33]. Since there have been no reports on the ultrastructure of R. minuta yeast cells, we aim to reveal the cell components dynamics during budding of this yeast with the use of modern methods of transmission electron microscopy.
MATERIALS AND METHODS
In present work we used culture of strain IFM 5756 from Culture Collection of the Research Center of Pathogenic Fungi (Chiba University, Japan).
The strain was cultivated for 24 h in YPD medium (1% yeast extract, 2% bactopepton and 2% glucose) on shaker at 30° C. They were observed on phase-contrast microscope (Olympus BH-2RFCA). For transmission electron microscopy (TEM), cells were collected by centrifugation and sandwiched between two copper grids. Then the samples were freeze-substituted in 2% osmium tetroxide/ acetone at - 80° C for 48 h and embedded in epoxy resin [2- 5]. The ultrathin sections (70 nm thick) were cut with a diamond knife and stained with uranyl acetate and lead citrate. Finally, the grids were covered with Super support films (Nisshin EM, Tokyo, Japan) and observed in JEM-1400 EX transmission electron microscope (JEOL, Tokyo, Japan).
RESULTS
Phase-contrast microscopy. The cells were spherical or ellipsoidal (3,5-6,5 x 2,5-4,5 ^m), and single or in small groups (Fig. 1 a).
Fig. 1. Phase-contrast (a) and transmission electron microscopy (b-l) of the in vitro growing R. minuta cells. B - bud; BS - budding scar; CW - cell wall; ER - endoplasmic reticulum; M - mitochondrium(ia), Mt - microtubule; N - nucleus; Nu - nucleolus; V -vacuole, Vs - vesicles. Scale bars: a - 10 |m, b - 1,5 |_im, c, d, f, h, m - 1 |m, e, i-l - 1 |m.
Fig. 2. Ultrastructure of in vitro growing R. minuta cells during (a-i) and after budding (j). Scale bars: a-g, i, j - 1 |m, h - 2 |m.
Fig. 3. Diagram showing the organelles migration during R. minuta budding: a - mother cell before budding; b-g - mother cell during budding; h - mother and daughter cell cells after separation.
Mother cells before budding. The interphase nucleus and large vacuole occupied the main cell volume, and were practically similar in size (Fig. 1 b, c). The nucleus was localized right next to the scar (Fig. 1 b, 3 a), was spherical (1,3 (m) or ellipsoidal (1,0x1,5 (m), contained moderate level of randomly distributed condensed chromatin (Fig. 1 b). The nucleolus was ellipsoidal (0,4x0,6 (m) and consisted of granular and fibrillar components (Fig. 1 c). There were numerous ribosomes on outer nuclear membrane.
The number of mitochondria varied from 3 to 5. They were spherical (0,4 (m) or ellipsoidal (0,3 x 0,5 (m). The mitochondria had more electron-dense matrix than cytosol. They had light lengthy dense cristae. Dense association of ribosomes on mitochondria were observed (Fig. 1e, g) in all growth phases.
The single large vacuole was localized in the basal part of the cell under the nucleus (Fig. 1 d, e, 3 a). The vacuole was filled with specific tightly localized thin fibrillar material.
There were no storage substances. The cytosol had moderate electron density (Fig. 1 b) and was rich in mono-and polyribosomes. Numerous (from 50 to 70 on median cell section) light (0,10 (m) and grey (0,06 (m) secretory vesicles were uniformly distributed on the cell periphery (Fig. 1 d). There were no Golgi cisterns. There were few short rough endoplasmic reticulum (ER) (Fig. 3 a), and localized close to the plasma membrane. There were no microbodies. The plasma membrane was closely adhered to the electron-transparent thin (0,16 (m) cell wall with a single budding scar (with mean diameter 0,6 (m, Fig. 1 b).
Budding. The bud formation first started in the scar zone by isodiametrical growth. At this stage, the moderate electron density cytosol, numerous free ribosomes and uniformly distributed grey secretory vesicles (Fig. 1 d, 3 c, d) were visible in bud. Then during bud growth several single small mitochondria, small vacuoles (from 1 to 3, Fig. 1 i, j, f, h, 2 c) filled with fibrillar material, a few short ER cisterns, and many secretory vesicles (Fig. 1 j, l) were observed. The sections on mitochondrial surfaces in the
mother cell and bud showed dense ribosomal distribution (Fig. 2 a, arrows). The small vacuoles appeared in mother cell (Fig. 1 i, j, 3 d) that later selectively migrated to the bud.
At early stage of budding the nucleus was localized near the wall in the middle part of the cell above the large vacuole (Fig. 1 d, 3 b). The sizes of nucleus and degree of chromatization were increased. Some changes in mitochondrial number and localization occurred. The number of mitochondria on median cells sections increased up to 8-10. They moved from the cell periphery to the nucleus outer membrane (Fig. 1 e, 3 b). The long (0,6-0,8 ^m, Fig. 1 e, g, f, h) and branching profile of those organelles appeared. As was the previous stage, the numerous ribosomes were located on the outer mitochondrial membrane (Fig. 1 e).
Several microfilaments (Fig. 1 i, arrow) and short microtubules (Fig. 1 k) were visible in the isthmus. They were connected with the plasma membrane and localized parallel to each other. Contrary to the microfilaments, the microtubules were localized deep in the cytosol and distantly from the plasma membrane. We also found the microtubules near mitochondria in mother cells and bud (Fig. 2 h). Sometimes there were the long mitochondrial profiles that migrated through the isthmus (Fig. 1 f, h), that may prove the presence of a giant mitochondrium.
At this stage the large vacuole was localized in the basal part of the mother cell, but the nucleus was in central. Several mitochondrial profiles (Fig. 2 b, 3 d) were arranged opposite the isthmus. The process of transition of the cytosol, free ribosomes, mitochondria, small vacuoles, secretory vesicles and cisterns of ER from the mother cell into the bud's content across the isthmus was visible. The same situation was observed inside of both cell types during the next stage of apical growth (Fig. 3 e-g). The bud shape became ellipsoidal (Fig. 2 a-d, 3 g). Near the nuclear membrane of the mother cell several long microtubules were observed before their transition to the bud cytoplasm.
The nucleus in the mother cell migrates from the central part to the apical area directly opposite the scar (Fig. 2 d, 3 f). Later, after the division, we observed one nucleus in the mother cell and the other in the bud (Fig. 2 e, f, g, 3 g). Their sizes were 1,4 ^m and the degree of chromatin condensation became higher. They contained small (0,4 x 0,8 ^m) moderate electron density nucleolus (Fig. 2 e) localized near the inner nuclear membrane. After the mitosis and before the septum formation, the process of migration of cytosol, free ribosomes, mitochondrium and other cell components from mother cell to the bud continued (Fig. 2 e, f).
The mother and daughter cells after se-paration. After the cell separation the ultrastructure and localization of organelles in the mother cell were the same as they were before budding (Fig. 3 h).
The daughter cell after separation underwent the isodiametrical growth which was accompanied by the formation of the vacuole in its basal part (Fig. 2 i, j, 3 h). During that period the spherical (1,4 ^m) nucleus was localized in the apical part of the cell opposite the scar. Its size increased and the degree of chromatization decreased. The synthesis of cytosol, free ribosomes, and proliferation of mitochondria, ER and peripheral secretory vesicles took place. At that period the long profiles (0,7-0,9 ^m) of mitochondria appeared near the nucleus (Fig. 2 i). After the growth completion, the daughter cell showed the
ultrastructure typical for mature mother cell.
DISCUSSION
For investigated R. minuta yeast cells the monopolar budding were typical [6]. The mother cell of R. minuta had one interphase nucleus and large vacuole, moderate number of mitochondria, rare cisterns of ER and numerous secretory vesicles. During budding of yeast the sizes of nucleus and nucleolus together with the level of chromatization increased. Similar data were obtained for in vitro growing cells at early stage of budding of C. albidus [4], M. pachydermatis [5] and C. laurentii [7]. The changes in topography of nucleus (i.e. the migration from central part to apical directly opposite the scar) of the budding mother cell were also typical for M. pachydermatis [5]. The other pattern of the nucleus migration was revealed for mother yeast cells of C. albidus [4] and C. laurentii [7], from the basal part to central-lateral and finally apical under the isthmus. The R. minuta, M. pachydermatis [5] and C. laurentii [7] mother cells at all the stages of budding did not differ between themselves in vacuolar content morphology and that significantly distinguished this species from C. albidus [4].
R. minuta budding was accompanied by the increase of number of mitochondria with the formation of one giant organelle proceeded according to two stages: 1) during budding in a mother cell; 2) after budding in a daughter cell. In a mother cell the formation of giant mitochondria was necessary for the synthesis of cytosol, free ribosomes and other cell components for the developing bud. In a daughter cell after budding the giant mitochondrium provides the same component synthesis but during its differentiation into mature mother cell. These morphogenetic peculiarities distinctly differ R. minuta from the species of other genus [C. albidus: 4; M. pachydermatis: 5]. The giant mitochondrium also developed during budding of yeast cells C. laurentii [7]. The aggregation of mitochondria was also revealed near the nucleus at early stages of budding in R. glutinis yeast cells [McCully E.K., Robinow C.F. J. Cell Sci. 1972; 11] and commonly was observed in R. rubra cells [Srivastava K.C., Smith D.G. Cytobios. 1979; 26]. Unusual morphological «alliance» between the outer membrane of mitochondrium and ribosomes may suggest that these topographical peculiarities considerably increased the degree of mitochondrial functional capability and activity in mother and daughter cells. We found similar aggregation of ribosomes with mitochondrium on Figures 1 b and 2 a with the images of mother and budding cells of other species - R. glutinis [McCully E.K., Robinow C.F. J. Cell Sci. 1972; 11]. It is interesting to note that in the yeast cells of M. pachydermatis [5], the number of mitochondria and their structure during budding were permanent that may be typical for other species of genus Malassezia.
The storage substances, Golgi cisterns and microbodies in R. minuta were absent. The absence of storage substances was also typical in C. albidus [4] and C. laurentii [7] cultivated for 24 h under the same conditions. Contrary,
for M. pachidermatis mother yeast cells [5] growing under similar conditions and time length, the presence of single large lipid inclusion was typical. Similar with R. minuta, the microbodies were absent in budding cells of C. albidus [4], M. pachydermmatis [5] and C. laurentii [7].
The analysis of R. minuta, C. albidus, M. pachydermatis and C. laurentii patterns of yeast budding demonstrated significant differences in organelles topography, quantity and quality in contents of budding mother cells depending on species. Topographical relations between the interphase nucleus and vacuoles during budding in two species of one genus Cryptococcus were identical, but differed between the species of different genus. The investigated species were divided into three distinct groups concordant with criteria of their functional ultrastructural activity. R. minuta with high level of fine structural activity during budding belongs to the first group C. albidus and C. laurentii with median level of organelle activity - to the second. In the cytosol of C. albidus, C. laurentii and R. minuta mother cells during early stages of bud formation we observed the proliferation of mitochondria with developed one giant organelle, which was concentrated around the nucleus. Only in R. minuta we revealed the repeated presence of a giant mitochondrium during the daughter cell development into the mother cell. This peculiarity in cell cycle accelerates the speed of cell growth and gives this species the specific priority. The third group, with lower level of budding activity was discovered in M. pachydermatis. For mother cells of this species before and during the budding the lower level of vacuolization, small number of mitochondria, presence of single cistern of ER, absence of secretory vesicles and presence of one big lipid inclusion opposite the budding scar was typical. During the process of bud morphogenesis the number of mitochondria, storage lipids and cisterns of ER was not increased contrary to the volume of cytosol and number of free ribosomes.
CONCLUSION
The revealed differences in the organellography during yeast cells budding may be very important for the phylogenetic investigations. Perhaps during the evolution the analyzed yeast species passed the process of «energization», so that the phylogenetic relations between them may be as follows: Malassezia ^Cryptococcus■ Rhodotorula. But may be a more «economical» pattern of budding, which we revealed for M. pachydermatis (transport of «ready» storage inclusion from the mother cell into the daughter's, the absence of mitochondrial proliferation and other peculiarities), phylogenetically was more advanced? The revealed differences in patterns of organelles transition during yeast cells budding of investigated species may indirectly demonstrate the differences in the «rapidity» of this process.
The subsequent investigations of other yeast species from one genus or from different genus of one family or different families will permit to solve the question on taxonomical importance of the revealed differences.
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Поступила в редакцию журнала 25.05.2017 Рецензент: Игнатьева С.М.
