Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.
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.
Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.Show MeSH
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Mentions: Mammalian cells encode scaffold proteins that interact with CN and substrates to target the phosphatase to specific subcellular compartments. For example, in neurons, AKAP79 localizes CN to L-type Ca2+ channels at the plasma membrane (Oliveria et al., 2007). In contrast, under standard growth conditions, yeast CN localized diffusely throughout the cell, as visualized in cells expressing the functional Cna1-3x green fluorescent protein (Cna1-3GFP) fusion protein from its endogenous locus (Figure 1A). Because CN is active during environmental stress, we also examined its distribution under a range of stress conditions. These analyses revealed that Cna1-3GFP distribution changed dramatically 10 min after addition of 1.25 M KCl to the growth medium, accumulating at foci throughout the cell body, the bud tip, and the bud neck. Cna2-3GFP, a functionally redundant catalytic subunit isoform, displayed similar localization changes under these conditions (Supplemental Figure S1A). This redistribution of CN was triggered by hypertonic shock, as NaCl (1.25 M), sorbitol (2 M), and KCl (1.25 M) all provoked the same response (Figure 1A), which occurred only during intense hyperosmotic stress (KCl > 0.75 M) and became more pervasive as osmotic strength increased (Supplemental Figure S1B). However, CN remained cytosolic in cells exposed either to 200 mM LiCl, a condition under which CN is required for survival that does not induce hyperosmotic shock (Figure 1A and Supplemental Figure S1C), or 50 mM CaCl2, which activates CN-dependent transcription without inducing osmotic stress (Figure 1A). Furthermore, hypertonic shock caused redistribution of Cna1-3GFP in cells treated with the CN inhibitor FK506 (Figure 1A) and in a CN-deficient mutant that lacks the regulatory subunit (cnb1; Supplemental Figure S1D). Therefore the cellular localization of CN changes specifically in response to intense hyperosmotic shock, and this redistribution in independent of CN activity. Surprisingly, the observed changes in CN localization did not depend on the HOG pathway and occurred robustly in hog1, pbs2, and ssk2 mutants exposed to hyperosmotic challenge (Supplemental Figure S1E). The requirement for intense osmotic shock to redistribute CN also distinguishes this response from HOG pathway activation, which occurs at much lower osmolarities (Schaber et al., 2010).
Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.