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Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.

Guiney EL, Goldman AR, Elias JE, Cyert MS - Mol. Biol. Cell (2014)

Bottom Line: By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells.This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion.We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.

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Calcineurin promotes actin repolarization during hyperosmotic stress. (A) Actin polarity during 4-h, 1.25 M KCl stress. Cells were pretreated for 30 min with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20), fixed at indicated time points, stained with rhodamine–phalloidin, and analyzed with CalMorph image analysis software as described in Materials and Methods. Cells with a bud/mother actin ratio (average fluorescence in bud)/(average fluorescence in mother cell) > 2 were scored as polarized. Data are from three independent experiments; N > 200 cells at each data point. †p < 10−9 from statistics as described in C. (B) Representative micrographs, stained for rhodamine–phalloidin. Scale bar, 5 μm. (C) Histograms of actin polarity measurements at 0, 120, and 150 min; vertical dotted line divides polarized and depolarized cells. Inset, Kruskal–Wallis mean ranks with 99% confidence intervals. †p < 10−9, Kruskal–Wallis rank sum test with Bonferroni correction.
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Figure 3: Calcineurin promotes actin repolarization during hyperosmotic stress. (A) Actin polarity during 4-h, 1.25 M KCl stress. Cells were pretreated for 30 min with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20), fixed at indicated time points, stained with rhodamine–phalloidin, and analyzed with CalMorph image analysis software as described in Materials and Methods. Cells with a bud/mother actin ratio (average fluorescence in bud)/(average fluorescence in mother cell) > 2 were scored as polarized. Data are from three independent experiments; N > 200 cells at each data point. †p < 10−9 from statistics as described in C. (B) Representative micrographs, stained for rhodamine–phalloidin. Scale bar, 5 μm. (C) Histograms of actin polarity measurements at 0, 120, and 150 min; vertical dotted line divides polarized and depolarized cells. Inset, Kruskal–Wallis mean ranks with 99% confidence intervals. †p < 10−9, Kruskal–Wallis rank sum test with Bonferroni correction.

Mentions: The depolarization and subsequent repolarization of the actin cytoskeleton is a prominent and acute cellular response to hyperosmotic shock (Chowdhury et al., 1992). CN localization to sites of polarized growth and partial colocalization with Abp1 suggested that the phosphatase might regulate the actin cytoskeleton during hyperosmotic shock. To test this idea, cells were exposed to 1.25 M KCl, fixed at 30-min intervals, stained for actin with rhodamine–phalloidin, and polarization quantified using image-analysis software (see Materials and Methods). Control cells were maximally depolarized (50%) 2 h after exposure to 1.25 M KCl, and polarity was largely restored by 4 h (Figure 3, A and B). Inactivation of CN with FK506 exacerbated depolarization at 120 and 150 min; however, as in control cells, polarity was reestablished by 4 h (Figure 3, A and B). Analyzing the distributions of cellular fluorescence ratios at 120 and 150 min after hyperosmotic shock showed that FK506 significantly decreased polarization compared with the control (Figure 3C; p < 10−9). Thus CN promotes repolarization of the actin cytoskeleton after hyperosmotic challenge.


Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.

Guiney EL, Goldman AR, Elias JE, Cyert MS - Mol. Biol. Cell (2014)

Calcineurin promotes actin repolarization during hyperosmotic stress. (A) Actin polarity during 4-h, 1.25 M KCl stress. Cells were pretreated for 30 min with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20), fixed at indicated time points, stained with rhodamine–phalloidin, and analyzed with CalMorph image analysis software as described in Materials and Methods. Cells with a bud/mother actin ratio (average fluorescence in bud)/(average fluorescence in mother cell) > 2 were scored as polarized. Data are from three independent experiments; N > 200 cells at each data point. †p < 10−9 from statistics as described in C. (B) Representative micrographs, stained for rhodamine–phalloidin. Scale bar, 5 μm. (C) Histograms of actin polarity measurements at 0, 120, and 150 min; vertical dotted line divides polarized and depolarized cells. Inset, Kruskal–Wallis mean ranks with 99% confidence intervals. †p < 10−9, Kruskal–Wallis rank sum test with Bonferroni correction.
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Related In: Results  -  Collection

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Figure 3: Calcineurin promotes actin repolarization during hyperosmotic stress. (A) Actin polarity during 4-h, 1.25 M KCl stress. Cells were pretreated for 30 min with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20), fixed at indicated time points, stained with rhodamine–phalloidin, and analyzed with CalMorph image analysis software as described in Materials and Methods. Cells with a bud/mother actin ratio (average fluorescence in bud)/(average fluorescence in mother cell) > 2 were scored as polarized. Data are from three independent experiments; N > 200 cells at each data point. †p < 10−9 from statistics as described in C. (B) Representative micrographs, stained for rhodamine–phalloidin. Scale bar, 5 μm. (C) Histograms of actin polarity measurements at 0, 120, and 150 min; vertical dotted line divides polarized and depolarized cells. Inset, Kruskal–Wallis mean ranks with 99% confidence intervals. †p < 10−9, Kruskal–Wallis rank sum test with Bonferroni correction.
Mentions: The depolarization and subsequent repolarization of the actin cytoskeleton is a prominent and acute cellular response to hyperosmotic shock (Chowdhury et al., 1992). CN localization to sites of polarized growth and partial colocalization with Abp1 suggested that the phosphatase might regulate the actin cytoskeleton during hyperosmotic shock. To test this idea, cells were exposed to 1.25 M KCl, fixed at 30-min intervals, stained for actin with rhodamine–phalloidin, and polarization quantified using image-analysis software (see Materials and Methods). Control cells were maximally depolarized (50%) 2 h after exposure to 1.25 M KCl, and polarity was largely restored by 4 h (Figure 3, A and B). Inactivation of CN with FK506 exacerbated depolarization at 120 and 150 min; however, as in control cells, polarity was reestablished by 4 h (Figure 3, A and B). Analyzing the distributions of cellular fluorescence ratios at 120 and 150 min after hyperosmotic shock showed that FK506 significantly decreased polarization compared with the control (Figure 3C; p < 10−9). Thus CN promotes repolarization of the actin cytoskeleton after hyperosmotic challenge.

Bottom Line: By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells.This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion.We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus