scholarly journals Vacuolar pH in yeast cells during pseudohyphal growth induced by nitrogen starvation

2017 ◽  
Author(s):  
Koji Makanae
Keyword(s):  
Intracellular Ph ◽  
Nitrogen Starvation ◽  
Vital Staining ◽  
Time Lapse ◽  
Neutral Red ◽  
Yeast Cells ◽  
Ph Indicator ◽  
Ph Gradients ◽  
Yeast Form ◽  
Pseudohyphal Growth

It has been reported that the intracellular pH of the budding yeast Saccharomyces cerevisiae is asymmetric between mother and daughter cells, and this asymmetry in pH underlies replicative aging and rejuvenation. S. cerevisiae growth morphology changes between the yeast form and pseudohyphal form, according to nutrient availability. A previous study reported that the replicative life span of pseudohyphal form cells is longer than that of yeast form cells in S. cerevisiae. However, the intracellular pH of pseudohyphal cells is unknown. To examine the intracellular pH of S. cerevisiae cells during pseudohyphal growth, vital staining was performed with neutral red, which is a pH indicator, of cells growing on nitrogen starvation (SLAD) medium. The results showed that the vacuoles of S. cerevisiae cells during pseudohyphal growth induced by nitrogen starvation formed polar pH gradients. The relationship between cell size and shape and the neutral red staining patterns suggested that the pH of cell vacuoles during pseudohyphal growth changed from uniformly near pH 6.8 to steep gradients of pH from vacuole ends along the long axis of the cell. The results of time-lapse imaging to examine vacuolar dynamics and neutral red staining suggested that the pH gradients were not formed simply by inheritance of vacuolar contents accompanying vacuolar movements.

scholarly journals Vacuolar pH in yeast cells during pseudohyphal growth induced by nitrogen starvation

2017 ◽  
Author(s):  
Koji Makanae
Keyword(s):  
Intracellular Ph ◽  
Nitrogen Starvation ◽  
Vital Staining ◽  
Time Lapse ◽  
Neutral Red ◽  
Yeast Cells ◽  
Ph Indicator ◽  
Ph Gradients ◽  
Yeast Form ◽  
Pseudohyphal Growth

It has been reported that the intracellular pH of the budding yeast Saccharomyces cerevisiae is asymmetric between mother and daughter cells, and this asymmetry in pH underlies replicative aging and rejuvenation. S. cerevisiae growth morphology changes between the yeast form and pseudohyphal form, according to nutrient availability. A previous study reported that the replicative life span of pseudohyphal form cells is longer than that of yeast form cells in S. cerevisiae. However, the intracellular pH of pseudohyphal cells is unknown. To examine the intracellular pH of S. cerevisiae cells during pseudohyphal growth, vital staining was performed with neutral red, which is a pH indicator, of cells growing on nitrogen starvation (SLAD) medium. The results showed that the vacuoles of S. cerevisiae cells during pseudohyphal growth induced by nitrogen starvation formed polar pH gradients. The relationship between cell size and shape and the neutral red staining patterns suggested that the pH of cell vacuoles during pseudohyphal growth changed from uniformly near pH 6.8 to steep gradients of pH from vacuole ends along the long axis of the cell. The results of time-lapse imaging to examine vacuolar dynamics and neutral red staining suggested that the pH gradients were not formed simply by inheritance of vacuolar contents accompanying vacuolar movements.

scholarly journals Green Fluorescent Protein as a Noninvasive Intracellular pH Indicator

1998 ◽  
Vol 74 (3) ◽  
pp. 1591-1599 ◽  
Author(s):  
Malea Kneen ◽  
Javier Farinas ◽  
Yuxin Li ◽  
A.S. Verkman
Keyword(s):  
Green Fluorescent Protein ◽  
Intracellular Ph ◽  
Fluorescent Protein ◽  
Ph Indicator ◽  
Green Fluorescent

Interdigital tissue chondrogenesis induced by surgical removal of the ectoderm in the embryonic chick leg bud

Development ◽  
1986 ◽  
Vol 94 (1) ◽  
pp. 231-244
Author(s):  
J. M. Hurle ◽  
Y. Gañan
Keyword(s):  
Cell Death ◽  
Vital Staining ◽  
Mesenchymal Cell ◽  
Neutral Red ◽  
Surgical Removal ◽  
Experimental Conditions ◽  
Local Inhibition ◽  
Dorsal Ectoderm ◽  
Interdigital Cell Death ◽  
Proximal Zone

In the present work, we have analysed the possible involvement of ectodermal tissue in the control of interdigital mesenchymal cell death. Two types of experiments were performed in the stages previous to the onset of interdigital cell death: (i) removal of the AER of the interdigit; (ii) removal of the dorsal ectoderm of the interdigit. After the operation embryos were sacrificed at 10–12h intervals and the leg buds were studied by whole-mount cartilage staining, vital staining with neutral red and scanning electron microscopy. Between stages 27 and 30, ridge removal caused a local inhibition of the growth of the interdigit. In a high percentage of the cases, ridge removal at these stages was followed 30–40 h later by the formation of ectopic nodules of cartilage in the interdigit. The incidence of ectopic cartilage formation was maximum at stage 29 (60%). In all cases, cell death took place on schedule although the intensity and extent of necrosis appeared diminished in relation to the intensity of inhibition of interdigital growth and to the presence of interdigital cartilages. Ridge removal at stage 31 did not cause inhibition of the growth of the interdigit and ectopic chondrogenesis was only detected in 3 out of 35 operated embryos. Dorsal ectoderm removal from the proximal zone of the interdigit at stage 29 caused the chondrogenesis of the proximal interdigital mesenchyme in 6 out of 18 operated embryos. The pattern of neutral red vital staining was consistent with these results revealing a partial inhibition of interdigital cell death in the proximal zone of the interdigit. It is proposed that under the present experimental conditions the mesenchymal cells are diverted from the death programme by a primary transformation into cartilage.

scholarly journals The TOR Signal Transduction Cascade Controls Cellular Differentiation in Response to Nutrients

2001 ◽  
Vol 12 (12) ◽  
pp. 4103-4113 ◽  
Author(s):  
N. Shane Cutler ◽  
Xuewen Pan ◽  
Joseph Heitman ◽  
Maria E. Cardenas
Keyword(s):  
Signal Transduction ◽  
Protein Kinases ◽  
Protein Phosphatase ◽  
Nutrient Sensing ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Growth Defect ◽  
Tor Pathway ◽  
Pseudohyphal Growth ◽  
Pseudohyphal Differentiation

Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiaeto humans. In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis. We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation. Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42–Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.

Intracellular pH measurements in gastric mucosa.

1979 ◽  
Vol 237 (1) ◽  
pp. E82
Author(s):  
S J Hersey
Keyword(s):  
Gastric Mucosa ◽  
Acid Secretion ◽  
Intracellular Ph ◽  
Ph Indicator ◽  
Active Secretion ◽  
Ph Measurements ◽  
Tubular Cells ◽  
A Value ◽  
Indicator Dye ◽  
Secretion Rates

Intracellular pH was measured in bullfrog gastric mucosa using a pH-indicator dye, bromthymol blue (BTB), with a spectrophotometric technique. Studies showed that BTB is taken up by the gastric mucosa and bound to intracellular components. The binding of BTB was shown to cause a shift in the pKa of the dye from the solution value of 6.95 to a value of 8.0. During the nonsecreting state, intracellular pH was estimated to be 7.4 (metiamide inhibition) or 7.1 (SCN inhibition). During active secretion of acid, intracellular pH increased with increasing secretion rates, reaching values in excess of pH 8. Using preparations from which the surface epithelial cells had been removed, it was shown that at least a portion of the alkaline response to stimulation occurs in the oxyntic or tubular cells. The results are interpreted in view of existing models for the chemical reaction involved in gastric acid secretion.

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

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