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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 membrane morphology via Inp53. (A) Representative images of inp∆∆∆ (LSY312) cells expressing GFP-2xPH(PLCδ) and high-copy INP53 (pRS426-INP53), vector only (pRS316), or low-copy INP53 or INP53ARAQAA (pRS316-INP53 or pRS316-INP53ARAQAA) were grown in dextrose, then treated with 1 μg/ml FK506 or vehicle for 2 h where indicated. Middle, membrane trace of GFP-2xPH(PLCδ) identified computationally (see Materials and Methods). Right, based on the computationally identified membrane region, we counted invaginations (marked in red) that extend further than 10 pixels (∼0.5 μm) into the cell (blue line). Scale bar, 5 μm. (B) Average number of large (0.5 μm) invaginations quantified from GFP-2xPH(PLCδ) membrane traces such as those shown in A. Error bars are SEM. ***p < 0.0001 for all pairwise comparisons except where marked; n.s, not significant; one-way ANOVA with Tukey's correction for multiple comparisons.
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Figure 6: Calcineurin regulates membrane morphology via Inp53. (A) Representative images of inp∆∆∆ (LSY312) cells expressing GFP-2xPH(PLCδ) and high-copy INP53 (pRS426-INP53), vector only (pRS316), or low-copy INP53 or INP53ARAQAA (pRS316-INP53 or pRS316-INP53ARAQAA) were grown in dextrose, then treated with 1 μg/ml FK506 or vehicle for 2 h where indicated. Middle, membrane trace of GFP-2xPH(PLCδ) identified computationally (see Materials and Methods). Right, based on the computationally identified membrane region, we counted invaginations (marked in red) that extend further than 10 pixels (∼0.5 μm) into the cell (blue line). Scale bar, 5 μm. (B) Average number of large (0.5 μm) invaginations quantified from GFP-2xPH(PLCδ) membrane traces such as those shown in A. Error bars are SEM. ***p < 0.0001 for all pairwise comparisons except where marked; n.s, not significant; one-way ANOVA with Tukey's correction for multiple comparisons.

Mentions: Inp53 exerts its effects on actin polarity by dephosphorylating PI(4,5)P2. This phospholipid is a specific component of the plasma membrane (Ooms et al., 2000; Stefan et al., 2002), and mutants lacking multiple synaptojanins display aberrant plasma membrane invaginations due to increased levels of PI(4,5)P2 and a defect in synap­tojanin-mediated membrane scission (Singer-Kruger et al., 1998; Sun et al., 2007). To determine whether regulation of Inp53 by CN affects plasma membrane morphology, we visualized the plasma membrane in inpΔΔΔ cells using the PI(4,5)P2-binding biosensor GFP-2xPH(PLCδ) (Stefan et al., 2002). In contrast to the normal, smooth plasma membranes observed in inpΔΔΔ cells expressing high-copy INP53, inpΔΔΔ cells expressing a vector, and thus depleted of INP53, formed massive, PI(4,5)P2-containing membrane deformations (Figure 6A). To quantify these structures, we computationally defined the membrane region of each cell using GFP-2xPH(PLCδ) intensity and counted regions that extended >10 pixels (∼0.5 um) into the cytoplasm (Figure 6, A and B). Synaptojanin-limited cells expressing low-copy INP53 had slightly more membrane invaginations per cell than the high-copy control, and inhibiting CN with FK506 exacerbated this defect. inpΔΔΔ cells expressing low copy INP53ARAQAA, which disrupts CN binding, displayed even more severe membrane deformation, which was further exacerbated by incubation with FK506. Thus analyses of both polarized actin distribution and plasma membrane morphology in cells expressing Inp53 as the sole synaptojanin revealed that CN must bind to and dephosphorylate Inp53 to achieve full synaptojanin function.


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

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

Calcineurin regulates membrane morphology via Inp53. (A) Representative images of inp∆∆∆ (LSY312) cells expressing GFP-2xPH(PLCδ) and high-copy INP53 (pRS426-INP53), vector only (pRS316), or low-copy INP53 or INP53ARAQAA (pRS316-INP53 or pRS316-INP53ARAQAA) were grown in dextrose, then treated with 1 μg/ml FK506 or vehicle for 2 h where indicated. Middle, membrane trace of GFP-2xPH(PLCδ) identified computationally (see Materials and Methods). Right, based on the computationally identified membrane region, we counted invaginations (marked in red) that extend further than 10 pixels (∼0.5 μm) into the cell (blue line). Scale bar, 5 μm. (B) Average number of large (0.5 μm) invaginations quantified from GFP-2xPH(PLCδ) membrane traces such as those shown in A. Error bars are SEM. ***p < 0.0001 for all pairwise comparisons except where marked; n.s, not significant; one-way ANOVA with Tukey's correction for multiple comparisons.
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Figure 6: Calcineurin regulates membrane morphology via Inp53. (A) Representative images of inp∆∆∆ (LSY312) cells expressing GFP-2xPH(PLCδ) and high-copy INP53 (pRS426-INP53), vector only (pRS316), or low-copy INP53 or INP53ARAQAA (pRS316-INP53 or pRS316-INP53ARAQAA) were grown in dextrose, then treated with 1 μg/ml FK506 or vehicle for 2 h where indicated. Middle, membrane trace of GFP-2xPH(PLCδ) identified computationally (see Materials and Methods). Right, based on the computationally identified membrane region, we counted invaginations (marked in red) that extend further than 10 pixels (∼0.5 μm) into the cell (blue line). Scale bar, 5 μm. (B) Average number of large (0.5 μm) invaginations quantified from GFP-2xPH(PLCδ) membrane traces such as those shown in A. Error bars are SEM. ***p < 0.0001 for all pairwise comparisons except where marked; n.s, not significant; one-way ANOVA with Tukey's correction for multiple comparisons.
Mentions: Inp53 exerts its effects on actin polarity by dephosphorylating PI(4,5)P2. This phospholipid is a specific component of the plasma membrane (Ooms et al., 2000; Stefan et al., 2002), and mutants lacking multiple synaptojanins display aberrant plasma membrane invaginations due to increased levels of PI(4,5)P2 and a defect in synap­tojanin-mediated membrane scission (Singer-Kruger et al., 1998; Sun et al., 2007). To determine whether regulation of Inp53 by CN affects plasma membrane morphology, we visualized the plasma membrane in inpΔΔΔ cells using the PI(4,5)P2-binding biosensor GFP-2xPH(PLCδ) (Stefan et al., 2002). In contrast to the normal, smooth plasma membranes observed in inpΔΔΔ cells expressing high-copy INP53, inpΔΔΔ cells expressing a vector, and thus depleted of INP53, formed massive, PI(4,5)P2-containing membrane deformations (Figure 6A). To quantify these structures, we computationally defined the membrane region of each cell using GFP-2xPH(PLCδ) intensity and counted regions that extended >10 pixels (∼0.5 um) into the cytoplasm (Figure 6, A and B). Synaptojanin-limited cells expressing low-copy INP53 had slightly more membrane invaginations per cell than the high-copy control, and inhibiting CN with FK506 exacerbated this defect. inpΔΔΔ cells expressing low copy INP53ARAQAA, which disrupts CN binding, displayed even more severe membrane deformation, which was further exacerbated by incubation with FK506. Thus analyses of both polarized actin distribution and plasma membrane morphology in cells expressing Inp53 as the sole synaptojanin revealed that CN must bind to and dephosphorylate Inp53 to achieve full synaptojanin function.

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