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Amiodarone induces cell wall channel formation in yeast Hansenula polymorpha.

Kalebina TS, Sokolov SS, Selyakh IO, Vanichkina DP, Severin FF - Springerplus (2015)

Bottom Line: Light microscopy shows that shortly after adding amiodarone, spherical structures, which can be stained with DNA binding dyes, form on the cell surface.Electron microphotographs show that amiodarone induces the formation of channels 50-80 nm in diameter in the cell wall that appear to be filled with intracellular material.Using fluorescent microscopy, we demonstrate MitoTracker-positive DNA-containing structures visibly extruded from the cells through these channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, Moscow, 119992 Russia.

ABSTRACT
The yeast cell wall is constantly remodeled to enable cell growth and division. In this study, we describe a novel type of cell wall modification. We report that the drug amiodarone induces rapid channel formation within the cell wall of the yeast Hansenula polymorpha. Light microscopy shows that shortly after adding amiodarone, spherical structures, which can be stained with DNA binding dyes, form on the cell surface. Electron microphotographs show that amiodarone induces the formation of channels 50-80 nm in diameter in the cell wall that appear to be filled with intracellular material. Using fluorescent microscopy, we demonstrate MitoTracker-positive DNA-containing structures visibly extruded from the cells through these channels. We speculate that the observed channel formation acts to enable the secretion of mitochondrial material from the cell under stressful conditions, thus enabling adaptive changes to the extracellular environment.

No MeSH data available.


Related in: MedlinePlus

Electron microphotographs of untreated Hansenula polymorpha cells (a) and cells treated with amiodarone (b–e). Electron-dense channels (black arrow) span the cell wall. G glucan layer, black arrowheads—mannoproteins. Electron-dense areas can be seen where the channels exit the cell (double black arrowheads). Bar length 0.2 μm
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Fig2: Electron microphotographs of untreated Hansenula polymorpha cells (a) and cells treated with amiodarone (b–e). Electron-dense channels (black arrow) span the cell wall. G glucan layer, black arrowheads—mannoproteins. Electron-dense areas can be seen where the channels exit the cell (double black arrowheads). Bar length 0.2 μm

Mentions: To see the effect of amiodarone on H. polymorpha, we treated cells grown in a liquid culture with varying concentrations of the drug and found that amiodarone at 80 μM concentration killed approximately 50 % of the cells. Using differential interference contrast (DIC) microscopy, we noticed that after addition of 80 μM amiodarone, cells formed cone-shaped protrusions and spherical structures at their surfaces (Fig. 1a, b) after 15 min of incubation with amiodarone. To determine the nature of these structures, we stained them with a number of fluorescent dyes. We found that both propidium iodide (PI) and 6′-diamidino-2-phenylindole (DAPI) (Fig. 1c, d) were accumulated by these spherical structures. Our observations suggested the intriguing possibility that DNA was being transported across the cell wall. We reasoned that dramatic changes must occur in cell wall morphology, and we used transmission electron microscopy (EM) to test this hypothesis. Electron microphotographs revealed that amiodarone triggered a number of structural changes in the cell wall: (1) the wall visibly thickened in an uneven way: (2) the electron-transparent glucan layer (marked G, Fig. 2a, b) thickened up to 200–400 nm, 2–4 times thicker than in the control; (3) the electron-dense external layer, supposedly of mannoprotein nature (Fig. 2b), becomes less dense and seemed thinner compared to a glucan layer (Fig. 2a–c). Crucially, during this process the cell did not change its size significantly (Fig. 1a, b). We also noticed that unusual structures were forming within the cell walls, which we called channels, because they spanned the cell wall (Fig. 2c–e). We estimate the diameter of the channels to be 50–80 nm. Interestingly, intracellular material appeared to be transferred across these channels (Fig. 2c–e).Fig. 1


Amiodarone induces cell wall channel formation in yeast Hansenula polymorpha.

Kalebina TS, Sokolov SS, Selyakh IO, Vanichkina DP, Severin FF - Springerplus (2015)

Electron microphotographs of untreated Hansenula polymorpha cells (a) and cells treated with amiodarone (b–e). Electron-dense channels (black arrow) span the cell wall. G glucan layer, black arrowheads—mannoproteins. Electron-dense areas can be seen where the channels exit the cell (double black arrowheads). Bar length 0.2 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549368&req=5

Fig2: Electron microphotographs of untreated Hansenula polymorpha cells (a) and cells treated with amiodarone (b–e). Electron-dense channels (black arrow) span the cell wall. G glucan layer, black arrowheads—mannoproteins. Electron-dense areas can be seen where the channels exit the cell (double black arrowheads). Bar length 0.2 μm
Mentions: To see the effect of amiodarone on H. polymorpha, we treated cells grown in a liquid culture with varying concentrations of the drug and found that amiodarone at 80 μM concentration killed approximately 50 % of the cells. Using differential interference contrast (DIC) microscopy, we noticed that after addition of 80 μM amiodarone, cells formed cone-shaped protrusions and spherical structures at their surfaces (Fig. 1a, b) after 15 min of incubation with amiodarone. To determine the nature of these structures, we stained them with a number of fluorescent dyes. We found that both propidium iodide (PI) and 6′-diamidino-2-phenylindole (DAPI) (Fig. 1c, d) were accumulated by these spherical structures. Our observations suggested the intriguing possibility that DNA was being transported across the cell wall. We reasoned that dramatic changes must occur in cell wall morphology, and we used transmission electron microscopy (EM) to test this hypothesis. Electron microphotographs revealed that amiodarone triggered a number of structural changes in the cell wall: (1) the wall visibly thickened in an uneven way: (2) the electron-transparent glucan layer (marked G, Fig. 2a, b) thickened up to 200–400 nm, 2–4 times thicker than in the control; (3) the electron-dense external layer, supposedly of mannoprotein nature (Fig. 2b), becomes less dense and seemed thinner compared to a glucan layer (Fig. 2a–c). Crucially, during this process the cell did not change its size significantly (Fig. 1a, b). We also noticed that unusual structures were forming within the cell walls, which we called channels, because they spanned the cell wall (Fig. 2c–e). We estimate the diameter of the channels to be 50–80 nm. Interestingly, intracellular material appeared to be transferred across these channels (Fig. 2c–e).Fig. 1

Bottom Line: Light microscopy shows that shortly after adding amiodarone, spherical structures, which can be stained with DNA binding dyes, form on the cell surface.Electron microphotographs show that amiodarone induces the formation of channels 50-80 nm in diameter in the cell wall that appear to be filled with intracellular material.Using fluorescent microscopy, we demonstrate MitoTracker-positive DNA-containing structures visibly extruded from the cells through these channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Faculty of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, Moscow, 119992 Russia.

ABSTRACT
The yeast cell wall is constantly remodeled to enable cell growth and division. In this study, we describe a novel type of cell wall modification. We report that the drug amiodarone induces rapid channel formation within the cell wall of the yeast Hansenula polymorpha. Light microscopy shows that shortly after adding amiodarone, spherical structures, which can be stained with DNA binding dyes, form on the cell surface. Electron microphotographs show that amiodarone induces the formation of channels 50-80 nm in diameter in the cell wall that appear to be filled with intracellular material. Using fluorescent microscopy, we demonstrate MitoTracker-positive DNA-containing structures visibly extruded from the cells through these channels. We speculate that the observed channel formation acts to enable the secretion of mitochondrial material from the cell under stressful conditions, thus enabling adaptive changes to the extracellular environment.

No MeSH data available.


Related in: MedlinePlus