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Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis.

Antonescu CN, Danuser G, Schmid SL - Mol. Biol. Cell (2010)

Bottom Line: We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production.DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically.Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

ABSTRACT
Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.

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Primary and secondary alcohols inhibit CME. (A) Tfn internalization was measured in BSC-1 cells treated with either 1- or 2-butanol (1%, vol/vol) for 20 min or left untreated (control), as indicated. Shown are the means ± SE of 3–5 independent experiments. * p < 0.05, relative to corresponding time point of the control condition. (B) PA levels were determined in cells either treated with 1% 1-butanol for 20 min or left untreated (control). Shown are the means ± SE of three independent experiments. (C) BSC-1 cells transfected with GFP-PH WT or PIP2-binding deficient mutant were treated as indicated, rounded-up and imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of at least 2 independent experiments. (D) BSC-1 cells stably expressing σ2-GFP were treated as indicated, fixed and then imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of 3 independent experiments. Scale bars 10 μm.
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Figure 1: Primary and secondary alcohols inhibit CME. (A) Tfn internalization was measured in BSC-1 cells treated with either 1- or 2-butanol (1%, vol/vol) for 20 min or left untreated (control), as indicated. Shown are the means ± SE of 3–5 independent experiments. * p < 0.05, relative to corresponding time point of the control condition. (B) PA levels were determined in cells either treated with 1% 1-butanol for 20 min or left untreated (control). Shown are the means ± SE of three independent experiments. (C) BSC-1 cells transfected with GFP-PH WT or PIP2-binding deficient mutant were treated as indicated, rounded-up and imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of at least 2 independent experiments. (D) BSC-1 cells stably expressing σ2-GFP were treated as indicated, fixed and then imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of 3 independent experiments. Scale bars 10 μm.

Mentions: To begin to assess the contribution of PLD to CME, BSC-1 cells were treated with primary alcohols, used routinely to inhibit PLD. As reported previously (Boucrot et al., 2006), Tfn internalization was potently inhibited by treatment with 1% 1-butanol (Figure 1A). Tfn internalization was also inhibited, albeit to a lesser extent, upon treatment with 1% 2-butanol, which is not expected to inhibit PLD (Figure 1A). Treatment of BSC-1 cells with lower concentrations of 1-butanol (0.1–0.25%) had little effect on Tfn internalization. This raised the concern that at least part of the inhibitory effect of 1% 1-butanol on CME might be independent of inhibition of PLD. We therefore directly measured the effect of 1-butanol on the cellular levels of PA. Surprisingly, treatment of BSC-1 cells with 1-butanol did not affect the levels of PA (Figure 1B); if anything there was a slight, albeit not significant increase in cellular PA levels. Of note, the assay used to measure PA is highly specific (Morita et al., 2009); therefore it is unlikely that phosphatidylbutanol formed in cells incubated with 1-butanol contributes to PA measured. Thus, the inhibitory effects of 1-butanol appear not to be related to inhibition of PLD-catalyzed PA production.


Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis.

Antonescu CN, Danuser G, Schmid SL - Mol. Biol. Cell (2010)

Primary and secondary alcohols inhibit CME. (A) Tfn internalization was measured in BSC-1 cells treated with either 1- or 2-butanol (1%, vol/vol) for 20 min or left untreated (control), as indicated. Shown are the means ± SE of 3–5 independent experiments. * p < 0.05, relative to corresponding time point of the control condition. (B) PA levels were determined in cells either treated with 1% 1-butanol for 20 min or left untreated (control). Shown are the means ± SE of three independent experiments. (C) BSC-1 cells transfected with GFP-PH WT or PIP2-binding deficient mutant were treated as indicated, rounded-up and imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of at least 2 independent experiments. (D) BSC-1 cells stably expressing σ2-GFP were treated as indicated, fixed and then imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of 3 independent experiments. Scale bars 10 μm.
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Related In: Results  -  Collection

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Figure 1: Primary and secondary alcohols inhibit CME. (A) Tfn internalization was measured in BSC-1 cells treated with either 1- or 2-butanol (1%, vol/vol) for 20 min or left untreated (control), as indicated. Shown are the means ± SE of 3–5 independent experiments. * p < 0.05, relative to corresponding time point of the control condition. (B) PA levels were determined in cells either treated with 1% 1-butanol for 20 min or left untreated (control). Shown are the means ± SE of three independent experiments. (C) BSC-1 cells transfected with GFP-PH WT or PIP2-binding deficient mutant were treated as indicated, rounded-up and imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of at least 2 independent experiments. (D) BSC-1 cells stably expressing σ2-GFP were treated as indicated, fixed and then imaged as described in Materials and Methods. Shown are fluorescence micrographs representative of 3 independent experiments. Scale bars 10 μm.
Mentions: To begin to assess the contribution of PLD to CME, BSC-1 cells were treated with primary alcohols, used routinely to inhibit PLD. As reported previously (Boucrot et al., 2006), Tfn internalization was potently inhibited by treatment with 1% 1-butanol (Figure 1A). Tfn internalization was also inhibited, albeit to a lesser extent, upon treatment with 1% 2-butanol, which is not expected to inhibit PLD (Figure 1A). Treatment of BSC-1 cells with lower concentrations of 1-butanol (0.1–0.25%) had little effect on Tfn internalization. This raised the concern that at least part of the inhibitory effect of 1% 1-butanol on CME might be independent of inhibition of PLD. We therefore directly measured the effect of 1-butanol on the cellular levels of PA. Surprisingly, treatment of BSC-1 cells with 1-butanol did not affect the levels of PA (Figure 1B); if anything there was a slight, albeit not significant increase in cellular PA levels. Of note, the assay used to measure PA is highly specific (Morita et al., 2009); therefore it is unlikely that phosphatidylbutanol formed in cells incubated with 1-butanol contributes to PA measured. Thus, the inhibitory effects of 1-butanol appear not to be related to inhibition of PLD-catalyzed PA production.

Bottom Line: We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production.DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically.Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

ABSTRACT
Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.

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