Limits...
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.

Show MeSH

Related in: MedlinePlus

Calcineurin regulates actin polarity via Inp53. (A) Inp53 growth rate (doublings per hour) during exponential growth in dextrose. (B) Representative micrographs of inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA stained with rhodamine–phalloidin. (C) Histograms of actin polarity measurements in inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA (pRS316-INP53 and pRS316- INP53ARAQAA) in dextrose without stress. N > 200 cells/condition; insets are Kruskal–Wallis mean ranks with 95% confidence interval; ***p < 0.0001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4325846&req=5

Figure 5: Calcineurin regulates actin polarity via Inp53. (A) Inp53 growth rate (doublings per hour) during exponential growth in dextrose. (B) Representative micrographs of inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA stained with rhodamine–phalloidin. (C) Histograms of actin polarity measurements in inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA (pRS316-INP53 and pRS316- INP53ARAQAA) in dextrose without stress. N > 200 cells/condition; insets are Kruskal–Wallis mean ranks with 95% confidence interval; ***p < 0.0001.

Mentions: To examine regulation of Inp53 by CN in the absence of partially redundant Inp51 and Inp52, we used an inp51 inp52 inp53 triple mutant; the viability of this strain (hereafter inp∆∆∆) was maintained by a galactose-regulated copy of INP53 that was stably integrated into the genome. As expected, inp∆∆∆ cells expressing a control vector were inviable when grown in dextrose-containing medium (Figure 5A). INP53 or INP53ARAQAA was introduced into inp∆∆∆ on either a low- or high-copy plasmid and provided the sole source of synaptojanin during growth on dextrose. In the absence of hyperosmotic stress, inp∆∆∆ cells expressing high-copy INP53 or INP53ARAQAA grew at a rate similar to wild-type cells and, like wild-type cells, showed no change in growth rate in the presence of FK506. In contrast, inp∆∆∆ cells expressing low-copy Inp53 were viable but grew slowly, indicating that synaptojanin activity was limiting. Expression of INP53ARAQAA at low-copy resulted in even slower growth, suggesting that interaction with CN was required for full activity of Inp53. FK506 further inhibited growth of cells expressing either INP53 or INP53ARAQAA, and neither strain grew appreciably in the presence of 1.25 M KCl (unpublished data). These data suggest that under synaptojanin-limiting conditions, CN is required for growth and that one of its essential functions is to positively regulate Inp53.


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

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

Calcineurin regulates actin polarity via Inp53. (A) Inp53 growth rate (doublings per hour) during exponential growth in dextrose. (B) Representative micrographs of inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA stained with rhodamine–phalloidin. (C) Histograms of actin polarity measurements in inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA (pRS316-INP53 and pRS316- INP53ARAQAA) in dextrose without stress. N > 200 cells/condition; insets are Kruskal–Wallis mean ranks with 95% confidence interval; ***p < 0.0001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325846&req=5

Figure 5: Calcineurin regulates actin polarity via Inp53. (A) Inp53 growth rate (doublings per hour) during exponential growth in dextrose. (B) Representative micrographs of inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA stained with rhodamine–phalloidin. (C) Histograms of actin polarity measurements in inpΔΔΔ cells expressing low-copy INP53 or INP53ARAQAA (pRS316-INP53 and pRS316- INP53ARAQAA) in dextrose without stress. N > 200 cells/condition; insets are Kruskal–Wallis mean ranks with 95% confidence interval; ***p < 0.0001.
Mentions: To examine regulation of Inp53 by CN in the absence of partially redundant Inp51 and Inp52, we used an inp51 inp52 inp53 triple mutant; the viability of this strain (hereafter inp∆∆∆) was maintained by a galactose-regulated copy of INP53 that was stably integrated into the genome. As expected, inp∆∆∆ cells expressing a control vector were inviable when grown in dextrose-containing medium (Figure 5A). INP53 or INP53ARAQAA was introduced into inp∆∆∆ on either a low- or high-copy plasmid and provided the sole source of synaptojanin during growth on dextrose. In the absence of hyperosmotic stress, inp∆∆∆ cells expressing high-copy INP53 or INP53ARAQAA grew at a rate similar to wild-type cells and, like wild-type cells, showed no change in growth rate in the presence of FK506. In contrast, inp∆∆∆ cells expressing low-copy Inp53 were viable but grew slowly, indicating that synaptojanin activity was limiting. Expression of INP53ARAQAA at low-copy resulted in even slower growth, suggesting that interaction with CN was required for full activity of Inp53. FK506 further inhibited growth of cells expressing either INP53 or INP53ARAQAA, and neither strain grew appreciably in the presence of 1.25 M KCl (unpublished data). These data suggest that under synaptojanin-limiting conditions, CN is required for growth and that one of its essential functions is to positively regulate Inp53.

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