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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 regulates PI(4,5)P2 distribution during osmotic shock. (A) Representative images of wild-type (BY4741) cells expressing GFP-2xPH(PLCδ). Cells were treated with 1 μg/ml FK506 or vehicle for 30 min; osmotic shock was induced with 1.25 M KCl for 10 min where indicated. (B) Membrane regions with >10% enriched GFP-2xPH(PLCδ) compared with adjacent cytoplasm are marked in green; see Materials and Methods. (C) The percentage of total membrane in each cell that was enriched for GFP-2xPH(PLCδ) was averaged and plotted. Error bars represent SEM. ***p < 0.0001; one-way ANOVA and Tukey's multiple comparison posttest. For each condition, N > 700 cells, from four independent experiments.
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Figure 7: Calcineurin regulates PI(4,5)P2 distribution during osmotic shock. (A) Representative images of wild-type (BY4741) cells expressing GFP-2xPH(PLCδ). Cells were treated with 1 μg/ml FK506 or vehicle for 30 min; osmotic shock was induced with 1.25 M KCl for 10 min where indicated. (B) Membrane regions with >10% enriched GFP-2xPH(PLCδ) compared with adjacent cytoplasm are marked in green; see Materials and Methods. (C) The percentage of total membrane in each cell that was enriched for GFP-2xPH(PLCδ) was averaged and plotted. Error bars represent SEM. ***p < 0.0001; one-way ANOVA and Tukey's multiple comparison posttest. For each condition, N > 700 cells, from four independent experiments.

Mentions: In unstressed WT cells, GFP-2xPH(PLCδ) was smoothly distributed throughout the plasma membrane and uniformly brighter than the adjacent cytoplasm (Figure 7A). In contrast, after a 10-min hyperosmotic shock, distribution of GFP-2xPH(PLCδ) at the membrane dramatically changed; fluorescence was confined to a few bright patches in each cell, which were separated by large regions depleted of GFP-2xPH(PLCδ), where fluorescence was not significantly higher than in the adjacent cytoplasm (Figure 7A). This change in GFP-2xPH(PLCδ) distribution was quantified by determining the percentage of total membrane area in each cell that displayed significant enrichment of GFP-2xPH(PLCδ) fluorescence relative to the adjacent cytoplasm (Figure 7B; see Materials and Methods). This analysis captured the uniform, bright GFP-2xPH(PLCδ) localization in unstressed cells: on average, >70% of the membrane showed enriched GFP-2xPH(PLCδ) fluorescence. In contrast, after hyperosmotic shock, only 17% of the membrane, on average, showed enriched GFP-2xPH(PLCδ) (Figure 7; p < 0.0001). These results document a major redistribution of PI(4,5)P2 into enriched and depleted membrane domains during osmotic shock, which may also underlie changes in membrane morphology previously described by electron microscopy (Dupont et al., 2010).


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

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

Calcineurin regulates PI(4,5)P2 distribution during osmotic shock. (A) Representative images of wild-type (BY4741) cells expressing GFP-2xPH(PLCδ). Cells were treated with 1 μg/ml FK506 or vehicle for 30 min; osmotic shock was induced with 1.25 M KCl for 10 min where indicated. (B) Membrane regions with >10% enriched GFP-2xPH(PLCδ) compared with adjacent cytoplasm are marked in green; see Materials and Methods. (C) The percentage of total membrane in each cell that was enriched for GFP-2xPH(PLCδ) was averaged and plotted. Error bars represent SEM. ***p < 0.0001; one-way ANOVA and Tukey's multiple comparison posttest. For each condition, N > 700 cells, from four independent experiments.
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Related In: Results  -  Collection

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Figure 7: Calcineurin regulates PI(4,5)P2 distribution during osmotic shock. (A) Representative images of wild-type (BY4741) cells expressing GFP-2xPH(PLCδ). Cells were treated with 1 μg/ml FK506 or vehicle for 30 min; osmotic shock was induced with 1.25 M KCl for 10 min where indicated. (B) Membrane regions with >10% enriched GFP-2xPH(PLCδ) compared with adjacent cytoplasm are marked in green; see Materials and Methods. (C) The percentage of total membrane in each cell that was enriched for GFP-2xPH(PLCδ) was averaged and plotted. Error bars represent SEM. ***p < 0.0001; one-way ANOVA and Tukey's multiple comparison posttest. For each condition, N > 700 cells, from four independent experiments.
Mentions: In unstressed WT cells, GFP-2xPH(PLCδ) was smoothly distributed throughout the plasma membrane and uniformly brighter than the adjacent cytoplasm (Figure 7A). In contrast, after a 10-min hyperosmotic shock, distribution of GFP-2xPH(PLCδ) at the membrane dramatically changed; fluorescence was confined to a few bright patches in each cell, which were separated by large regions depleted of GFP-2xPH(PLCδ), where fluorescence was not significantly higher than in the adjacent cytoplasm (Figure 7A). This change in GFP-2xPH(PLCδ) distribution was quantified by determining the percentage of total membrane area in each cell that displayed significant enrichment of GFP-2xPH(PLCδ) fluorescence relative to the adjacent cytoplasm (Figure 7B; see Materials and Methods). This analysis captured the uniform, bright GFP-2xPH(PLCδ) localization in unstressed cells: on average, >70% of the membrane showed enriched GFP-2xPH(PLCδ) fluorescence. In contrast, after hyperosmotic shock, only 17% of the membrane, on average, showed enriched GFP-2xPH(PLCδ) (Figure 7; p < 0.0001). These results document a major redistribution of PI(4,5)P2 into enriched and depleted membrane domains during osmotic shock, which may also underlie changes in membrane morphology previously described by electron microscopy (Dupont et al., 2010).

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