scholarly journals Echinocandin Drugs Induce Differential Effects in Cytokinesis Progression and Cell Integrity

2021 ◽  
Vol 14 (12) ◽  
pp. 1332
Author(s):  
Natalia Yagüe ◽  
Laura Gómez-Delgado ◽  
M. Ángeles Curto ◽  
Vanessa S. D. Carvalho ◽  
M. Belén Moreno ◽  
...  
Keyword(s):  
Cell Death ◽  
Fluorescence Microscopy ◽  
Fission Yeast ◽  
Mechanism Of Action ◽  
Time Lapse ◽  
Cell Lysis ◽  
Cell Integrity ◽  
Wild Type ◽  
Fungicidal Effect ◽  
Sublethal Concentrations

Fission yeast contains three essential β(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of bgs1+ and bgs3+. Thus, Bgs1 and Bgs3 functions seem to be unaffected by those GS inhibitors. To learn more about echinocandins’ mechanism of action and resistance, cytokinesis progression and cell death were examined by time-lapse fluorescence microscopy in WT and pbr1-8 cells at the start of treatment with sublethal and lethal concentrations of anidulafungin, caspofungin, and micafungin. In WT, sublethal concentrations of the three drugs caused abundant cell death that was either suppressed (anidulafungin and micafungin) or greatly reduced (caspofungin) in pbr1-8 cells. Interestingly, the lethal concentrations induced differential phenotypes depending on the echinocandin used. Anidulafungin and caspofungin were mostly fungistatic, heavily impairing cytokinesis progression in both WT and pbr1-8. As with sublethal concentrations, lethal concentrations of micafungin were primarily fungicidal in WT cells, causing cell lysis without impairing cytokinesis. The lytic phenotype was suppressed again in pbr1-8 cells. Our results suggest that micafungin always exerts its fungicidal effect by solely inhibiting Bgs4. In contrast, lethal concentrations of anidulafungin and caspofungin cause an early cytokinesis arrest, probably by the combined inhibition of several GSs.

The size control of fission yeast revisited

1996 ◽  
Vol 109 (12) ◽  
pp. 2947-2957 ◽  
Author(s):  
A. Sveiczer ◽  
B. Novak ◽  
J.M. Mitchison
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Size Control ◽  
Time Lapse ◽  
Critical Threshold ◽  
Wild Type ◽  
Linear Rate ◽  
Cycle Times ◽  
Threshold Size ◽  
Histone Kinase

An analysis was made of cell length and cycle time in time-lapse films of the fission yeast Schizosaccharomyces pombe using wild-type (WT) cells and those of various mutants. The more important conclusions about ‘size controls’ are: (1) there is a marker in G2 in WT cells provided by a rate change point (RCP) where the linear rate of length growth increases by approximately 30%. The period before this RCP is dependent on size and can be called a ‘sizer’. The period after the RCP is nearly independent of size and can be called a ‘timer’. The achievement of a critical threshold size is at or near the RCP which is on average at about 0.3 of the cycle (halfway through G2). This is much earlier than was previously believed. (2) The RCP is at about the time when H1 histone kinase activity and the B type cyclin cdc13 start to rise in preparation for mitosis. The RCP is also associated with other metabolic changes. (3) In wee1 mutants, the mitotic size control is replaced by a G1/S size control which is as strong as the mitotic control. As in WT cells, there is a sizer which precedes the RCP followed by a timer but the RCP is at about the G1/S boundary and has a larger increase (approximately 100%) in rate. (4) cdc25 is not an essential part of the size control at mitosis or at the G1/S boundary. (5) Three further situations have been examined in which the mitotic size control has been abolished. First, induction synchronisation by block and release of cdc2 and cdc10. In the largest oversize-cells which are produced, the RCP is pushed back to the beginning of the cycle. There is no sizer period but only a timer. Second, when both the antagonists wee1 and cdc25 are absent in the double mutant wee1-50 cdc25 delta. In this interesting situation there is apparently no mitotic size control and the cycle times are quantised. Third, in rum1 delta wee1-50 where the normal long G1 in wee1 is much reduced, there is probably no size control either in G1 or in G2 causing a continuous shortening of division length from cycle to cycle.

Human p53 inhibits growth in Schizosaccharomyces pombe

1992 ◽  
Vol 12 (4) ◽  
pp. 1405-1411
Author(s):  
J R Bischoff ◽  
D Casso ◽  
D Beach
Keyword(s):  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Amino Acid Residue ◽  
Mechanism Of Action ◽  
Mammalian Cells ◽  
Simple System ◽  
Wild Type ◽  
Dominant Mutation ◽  
Mutant Forms

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.

scholarly journals The Antimicrobial Peptide Polyphemusin Localizes to the Cytoplasm of Escherichia coli following Treatment

2006 ◽  
Vol 50 (4) ◽  
pp. 1522-1524 ◽  
Author(s):  
Jon-Paul S. Powers ◽  
Morgan M. Martin ◽  
Danika L. Goosney ◽  
Robert E. W. Hancock
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Fluorescence Microscopy ◽  
Antimicrobial Peptide ◽  
Mechanism Of Action ◽  
Horseshoe Crab ◽  
Membrane Damage ◽  
Wild Type ◽  
High Antimicrobial Activity ◽  
Confocal Fluorescence

ABSTRACT The horseshoe crab peptide polyphemusin I possesses high antimicrobial activity, but its mechanism of action is as yet not well defined. Using a biotin-labeled polyphemusin I analogue and confocal fluorescence microscopy, we showed that the peptide accumulates in the cytoplasm of wild-type Escherichia coli within 30 min after addition without causing substantial membrane damage.

scholarly journals Palytoxin-induced cell death cascade in bovine aortic endothelial cells

2006 ◽  
Vol 291 (4) ◽  
pp. C657-C667 ◽  
Author(s):  
William P. Schilling ◽  
Deborah Snyder ◽  
William G. Sinkins ◽  
Mark Estacion
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Cell Lysis ◽  
Monovalent Cation ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Cell Death Cascade ◽  
Aortic Endothelial

The plasmalemmal Na+-K+-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca2+. However, the ability of PTX to directly increase cytosolic free Ca2+ concentration ([Ca2+]i) via Na+ pump channels and to initiate Ca2+ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca2+]i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca2+]i that was dependent on extracellular Ca2+. The increase in [Ca2+]i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC50 < 1 μM. The elevation in [Ca2+]i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca2+]i occurs via the Na+ pump. Subsequent to the rise in [Ca2+]i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca2+ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca2+ overload.

scholarly journals Human p53 inhibits growth in Schizosaccharomyces pombe.

1992 ◽  
Vol 12 (4) ◽  
pp. 1405-1411 ◽  
Author(s):  
J R Bischoff ◽  
D Casso ◽  
D Beach
Keyword(s):  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Amino Acid Residue ◽  
Mechanism Of Action ◽  
Mammalian Cells ◽  
Simple System ◽  
Wild Type ◽  
Dominant Mutation ◽  
Mutant Forms

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.

Blockade of maitotoxin-induced endothelial cell lysis by glycine and l-alanine

2003 ◽  
Vol 284 (4) ◽  
pp. C1006-C1020 ◽  
Author(s):  
Mark Estacion ◽  
Justin S. Weinberg ◽  
William G. Sinkins ◽  
William P. Schilling
Keyword(s):  
Cell Death ◽  
Endothelial Cell ◽  
Surface Membrane ◽  
Molecular Size ◽  
Time Lapse ◽  
Cell Lysis ◽  
Second Phase ◽  
Dye Uptake ◽  
Aortic Endothelial Cells ◽  
Bleb Formation

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs) is a model for oncotic/necrotic cell death. The cascade is initiated by an increase in cytosolic free Ca2+ concentration ([Ca2+]i), which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects activation of large pores and correlates with surface membrane bleb formation; the second phase reflects cell lysis. In the present study, the effect of the cytoprotective amino acid glycine was examined. Glycine had no effect on MTX-induced change in [Ca2+]i or on the first phase of vital dye uptake but produced a concentration-dependent (EC50 ∼1 mM) inhibition of the second phase of dye uptake. No cytoprotective effect was observed with l-valine, l-proline, or d-alanine, whereas l-alanine was equieffective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation. To test the hypothesis that glycine specifically blocks formation of a lytic “pore,” the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in size from 27 to 162 kDa was examined using time-lapse videomicroscopy. MTX-induced loss of GFP was rapid, correlated with the second phase of dye uptake, and was relatively independent of molecular size. The MTX-induced loss of GFP from BAECs was completely blocked by glycine. The data suggest that the second “lytic” phase of MTX-induced endothelial cell death reflects formation of a novel permeability pathway that allows macromolecules such as GFP or LDH to escape, yet can be prevented by the cytoprotective agents glycine andl-alanine.

Pattern of end growth of the fission yeast Schizosaccharomyces pombe

1990 ◽  
Vol 36 (6) ◽  
pp. 390-394 ◽  
Author(s):  
Hisao Miyata ◽  
Machiko Miyata ◽  
Byron F. Johnson
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Schizosaccharomyces Pombe ◽  
Key Words ◽  
Fission Yeast ◽  
Doubling Time ◽  
Time Lapse ◽  
Critical Value ◽  
Wild Type ◽  
Time Lapse Photomicrography

The patterns of end growth of individual cells of Schizosaccharomyces pombe, wild-type cells (strain 972 h−), cells exposed to 8 mM hydroxyurea, and cdc mutants (cdc11-123 and cdc2-33), were investigated by time-lapse photomicrography. It was reconfirmed that there are three patterns of end growth: cells growing at the old end, at the new end, and at both ends from the beginning of the cell cycle. Cells that initiated growth at the old (new) end increased their growth rate at the new (old) end and became constant in their growth rate at the old (new) end when cells had their growth rate higher than a critical value: 0.08, 0.09, 0.08, and 0.11 μm/min in wild-type cells, cells exposed to hydroxyurea, cdc11-123 cells, and cdc2-33 cells, respectively. The critical value is proportional to the doubling time in length. Key words: extension, growth, fission yeast.

Patterned epidermal cell death in wild-type and segment polarity mutant Drosophila embryos

Development ◽  
1998 ◽  
Vol 125 (17) ◽  
pp. 3427-3436 ◽  
Author(s):  
T.M. Pazdera ◽  
P. Janardhan ◽  
J.S. Minden
Keyword(s):  
Cell Death ◽  
Embryonic Development ◽  
Epidermal Cell ◽  
Time Lapse ◽  
Wild Type ◽  
Segment Polarity ◽  
Genetic Perturbations ◽  
Mutant Phenotypes ◽  
Cell Death Pattern ◽  
Drosophila Embryos

Programmed cell death plays an essential role in the normal embryonic development of Drosophila melanogaster. One region of the embryo where cell death occurs, but has not been studied in detail, is the abdominal epidermis. Because cell death is a fleeting process, we have used time-lapse, fluorescence microscopy to map epidermal apoptosis throughout embryonic development. Cell death occurs in a stereotypically striped pattern near both sides of the segment border and to a lesser extent in the middle of the segment. This map of wild-type cell death was used to determine how cell death patterns change in response to genetic perturbations that affect epidermal patterning. Previous studies have suggested that segment polarity mutant phenotypes are partially the result of increased cell death. Mutations in wingless, armadillo, and gooseberry led to dramatic increases in apoptosis in the anterior of the segment while a naked mutation resulted in a dramatic increase in the death of engrailed cells in the posterior of the segment. These results show that segment polarity gene expression is necessary for the survival of specific rows of epidermal cells and may provide insight into the establishment of the wild-type epidermal cell death pattern.

Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomyces pombe: video fluorescence microscopy of synchronously growing wild-type and cold-sensitive cdc mutants by using a DNA-binding fluorescent probe

1981 ◽  
Vol 52 (1) ◽  
pp. 271-287
Author(s):  
T. Toda ◽  
M. Yamamoto ◽  
M. Yanagida
Keyword(s):  
Dna Binding ◽  
Fluorescence Microscopy ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Fluorescent Probe ◽  
Temperature Shift ◽  
Nuclear Chromatin ◽  
Wild Type ◽  
Chromatin Region ◽  
3 Dimensional

Video-connected fluorescence microscopy was introduced to study the yeast nuclear chromatin region. It was defined as the nuclear area where a DNA-binding fluorescent probe 4′,6-diamidino-2-phenylindole specifically bound and fluoresced. The 3-dimensional feature of the mitotic chromatin region was deduced by analysing the successive video images of a cell viewed at different angles. By investigating synchronous culture of the wild-type fission yeast Schizosaccharomyces pombe, we found sequential structural alterations in the chromatin region during mitosis. The steps found include the compaction of the chromatin region from the regular hemispherical form, the formation of a U-shaped intermediate and the rapid segregation into 2 daughter hemispherical forms. Six cs cdc mutants, apparently blocked in mitosis, were observed by fluorescence microscopy. Under the restrictive conditions their chromatin regions exhibited either hemispherical, compact, disk-like, U-shaped or partially segregated chromatin regions. Two mutants showed anomalous nuclear locations. The results of the temperature shift-up experiments of the highly reversible KM52 and KM108 strains supported the above scheme of sequential alterations in the chromatin region.

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