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Phosphoinositide kinase signaling controls ER-PM cross-talk.

Omnus DJ, Manford AG, Bader JM, Emr SD, Stefan CJ - Mol. Biol. Cell (2016)

Bottom Line: However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress.We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca(2+)-regulated lipid biogenesis, upon plasma membrane (PM) stress.Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

ER-PM junctions regulate sphingolipid synthesis. (A) The yeast biosynthetic pathway for sphingolipid synthesis in the ER and Golgi network. (B) Sphingolipid synthesis in the ER and Golgi complex protects PM integrity during heat stress. Wild-type, lcb1ts, lac1 lag1ts, and aur1ts cells incubated at 26 (white bars) or 40°C for 2 h (black bars) were stained with propidium iodide and analyzed by flow cytometry. (C) Analysis of sphingolipid synthesis in wild-type and Δtether cells. Wild-type and Δtether cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and the complex sphingolipids IPCs, MIPC, and M(IP)2C are indicated. An unknown lipid, X, was also observed. The hatched areas point out reduced ceramides and MIPC in the Δtether mutant cells. (D) Synthesis of ceramides in wild-type and Δtether cells. Results shown here are from a long exposure from an independent experiment to better visualize ceramides. As in Figure 2C, cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and an unknown lipid, X, are indicated. (E) Quantitation of ceramide synthesis in wild-type and Δtether cells normalized to wild type at 26°C. The data represent the mean ± SD from three independent experiments.
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Figure 2: ER-PM junctions regulate sphingolipid synthesis. (A) The yeast biosynthetic pathway for sphingolipid synthesis in the ER and Golgi network. (B) Sphingolipid synthesis in the ER and Golgi complex protects PM integrity during heat stress. Wild-type, lcb1ts, lac1 lag1ts, and aur1ts cells incubated at 26 (white bars) or 40°C for 2 h (black bars) were stained with propidium iodide and analyzed by flow cytometry. (C) Analysis of sphingolipid synthesis in wild-type and Δtether cells. Wild-type and Δtether cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and the complex sphingolipids IPCs, MIPC, and M(IP)2C are indicated. An unknown lipid, X, was also observed. The hatched areas point out reduced ceramides and MIPC in the Δtether mutant cells. (D) Synthesis of ceramides in wild-type and Δtether cells. Results shown here are from a long exposure from an independent experiment to better visualize ceramides. As in Figure 2C, cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and an unknown lipid, X, are indicated. (E) Quantitation of ceramide synthesis in wild-type and Δtether cells normalized to wild type at 26°C. The data represent the mean ± SD from three independent experiments.

Mentions: Cells up-regulate de novo sphingolipid synthesis in the ER during heat-induced membrane stress (Tabuchi et al., 2006; Cowart and Hannun, 2007; Cowart and Obeid, 2007; Sun et al., 2012; Muir et al., 2014). We examined whether de novo sphingolipid synthesis is necessary to maintain PM integrity upon heat stress and whether ER-PM cross-talk is involved in this membrane stress response pathway. Sphingolipid synthesis is initiated in the ER, where long- chain sphingoid bases (LCBs) and ceramides are generated by the ER-localized serine palmitolyltransferase (SPT) and ceramide synthases, respectively (Figure 2A). We first examined whether de novo sphingolipid synthesis was necessary to maintain PM integrity upon heat stress. At 40°C, >25% of lcb1ts mutant cells (with impaired SPT activity) and 12% of lac1 lag1ts mutant cells (with impaired ceramide synthase activity) stained with propidium iodide (20- and 10-fold greater than wild-type cells, respectively; Figure 2B). In addition, 15% of aur1ts cells impaired in complex sphingolipid synthesis in the Golgi network displayed PM integrity defects upon shift to 40°C (Figure 2, A and B). Thus sphingolipid synthesis in the ER and Golgi compartments is needed for PM integrity during heat stress.


Phosphoinositide kinase signaling controls ER-PM cross-talk.

Omnus DJ, Manford AG, Bader JM, Emr SD, Stefan CJ - Mol. Biol. Cell (2016)

ER-PM junctions regulate sphingolipid synthesis. (A) The yeast biosynthetic pathway for sphingolipid synthesis in the ER and Golgi network. (B) Sphingolipid synthesis in the ER and Golgi complex protects PM integrity during heat stress. Wild-type, lcb1ts, lac1 lag1ts, and aur1ts cells incubated at 26 (white bars) or 40°C for 2 h (black bars) were stained with propidium iodide and analyzed by flow cytometry. (C) Analysis of sphingolipid synthesis in wild-type and Δtether cells. Wild-type and Δtether cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and the complex sphingolipids IPCs, MIPC, and M(IP)2C are indicated. An unknown lipid, X, was also observed. The hatched areas point out reduced ceramides and MIPC in the Δtether mutant cells. (D) Synthesis of ceramides in wild-type and Δtether cells. Results shown here are from a long exposure from an independent experiment to better visualize ceramides. As in Figure 2C, cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and an unknown lipid, X, are indicated. (E) Quantitation of ceramide synthesis in wild-type and Δtether cells normalized to wild type at 26°C. The data represent the mean ± SD from three independent experiments.
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Related In: Results  -  Collection

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Figure 2: ER-PM junctions regulate sphingolipid synthesis. (A) The yeast biosynthetic pathway for sphingolipid synthesis in the ER and Golgi network. (B) Sphingolipid synthesis in the ER and Golgi complex protects PM integrity during heat stress. Wild-type, lcb1ts, lac1 lag1ts, and aur1ts cells incubated at 26 (white bars) or 40°C for 2 h (black bars) were stained with propidium iodide and analyzed by flow cytometry. (C) Analysis of sphingolipid synthesis in wild-type and Δtether cells. Wild-type and Δtether cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and the complex sphingolipids IPCs, MIPC, and M(IP)2C are indicated. An unknown lipid, X, was also observed. The hatched areas point out reduced ceramides and MIPC in the Δtether mutant cells. (D) Synthesis of ceramides in wild-type and Δtether cells. Results shown here are from a long exposure from an independent experiment to better visualize ceramides. As in Figure 2C, cells were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. Ceramides (Cer) and an unknown lipid, X, are indicated. (E) Quantitation of ceramide synthesis in wild-type and Δtether cells normalized to wild type at 26°C. The data represent the mean ± SD from three independent experiments.
Mentions: Cells up-regulate de novo sphingolipid synthesis in the ER during heat-induced membrane stress (Tabuchi et al., 2006; Cowart and Hannun, 2007; Cowart and Obeid, 2007; Sun et al., 2012; Muir et al., 2014). We examined whether de novo sphingolipid synthesis is necessary to maintain PM integrity upon heat stress and whether ER-PM cross-talk is involved in this membrane stress response pathway. Sphingolipid synthesis is initiated in the ER, where long- chain sphingoid bases (LCBs) and ceramides are generated by the ER-localized serine palmitolyltransferase (SPT) and ceramide synthases, respectively (Figure 2A). We first examined whether de novo sphingolipid synthesis was necessary to maintain PM integrity upon heat stress. At 40°C, >25% of lcb1ts mutant cells (with impaired SPT activity) and 12% of lac1 lag1ts mutant cells (with impaired ceramide synthase activity) stained with propidium iodide (20- and 10-fold greater than wild-type cells, respectively; Figure 2B). In addition, 15% of aur1ts cells impaired in complex sphingolipid synthesis in the Golgi network displayed PM integrity defects upon shift to 40°C (Figure 2, A and B). Thus sphingolipid synthesis in the ER and Golgi compartments is needed for PM integrity during heat stress.

Bottom Line: However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress.We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca(2+)-regulated lipid biogenesis, upon plasma membrane (PM) stress.Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom.

No MeSH data available.


Related in: MedlinePlus