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Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2).

Hammond GR, Schiavo G, Irvine RF - Biochem. J. (2009)

Bottom Line: PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2).Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus.In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK. gruh2@cam.ac.uk

ABSTRACT
PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2). Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

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Competition for plasma membrane PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins were included at 10 μM with the primary anti-PtdIns4P or anti-PtdIns(4,5)P2 antibodies applied to COS-7 cells. Staining conditions are as in Figure 7. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. DAPI stained nuclei are in blue indicating the presence of cells where lipid staining is absent. (b) Quantification of staining (n=126 cells for anti-PtdIns4P antibody and 123 cells for anti-PtdIns(4,5)P2 antibody). See the Experimental section for details. ***P<0.001.
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Figure 8: Competition for plasma membrane PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins were included at 10 μM with the primary anti-PtdIns4P or anti-PtdIns(4,5)P2 antibodies applied to COS-7 cells. Staining conditions are as in Figure 7. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. DAPI stained nuclei are in blue indicating the presence of cells where lipid staining is absent. (b) Quantification of staining (n=126 cells for anti-PtdIns4P antibody and 123 cells for anti-PtdIns(4,5)P2 antibody). See the Experimental section for details. ***P<0.001.

Mentions: The specificity of the staining was again assessed by sequestration of endogenous PtdIns4P with GST–PH-FAPP1. As seen for Golgi staining, this completely abolished staining, whereas GST alone had no effect (Figure 8). However, the PtdIns(4,5)P2-sequestering PH domain from PLCδ1 produced a near ablation of staining (Figure 8). This is in marked contrast with parallel experiments using anti-PtdIns(4,5)P2 antibody, where only GST–PH-PLCδ1 was able to abolish staining, consistent with our earlier report [29]. Given the specific competition of PtdIns4P staining by exogenous PtdIns4P compared with PtdIns(4,5)P2 (Figure 1), it seems unlikely that this result reflects reactivity of anti-PtdIns4P antibody with plasma membrane PtdIns(4,5)P2. As argued above, it may simply reflect binding of high concentrations of GST–PH-PLCδ1 to plasma membrane PtdIns4P. However, given the enrichment of PtdIns(4,5)P2 at the plasma membrane, cross-reactivity was more of a concern than for Golgi staining, so we conceived further experiments to eliminate this possibility.


Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2).

Hammond GR, Schiavo G, Irvine RF - Biochem. J. (2009)

Competition for plasma membrane PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins were included at 10 μM with the primary anti-PtdIns4P or anti-PtdIns(4,5)P2 antibodies applied to COS-7 cells. Staining conditions are as in Figure 7. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. DAPI stained nuclei are in blue indicating the presence of cells where lipid staining is absent. (b) Quantification of staining (n=126 cells for anti-PtdIns4P antibody and 123 cells for anti-PtdIns(4,5)P2 antibody). See the Experimental section for details. ***P<0.001.
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Related In: Results  -  Collection

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Show All Figures
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Figure 8: Competition for plasma membrane PtdIns4P staining by PH-FAPP1(a) The indicated GST-tagged fusion proteins were included at 10 μM with the primary anti-PtdIns4P or anti-PtdIns(4,5)P2 antibodies applied to COS-7 cells. Staining conditions are as in Figure 7. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. DAPI stained nuclei are in blue indicating the presence of cells where lipid staining is absent. (b) Quantification of staining (n=126 cells for anti-PtdIns4P antibody and 123 cells for anti-PtdIns(4,5)P2 antibody). See the Experimental section for details. ***P<0.001.
Mentions: The specificity of the staining was again assessed by sequestration of endogenous PtdIns4P with GST–PH-FAPP1. As seen for Golgi staining, this completely abolished staining, whereas GST alone had no effect (Figure 8). However, the PtdIns(4,5)P2-sequestering PH domain from PLCδ1 produced a near ablation of staining (Figure 8). This is in marked contrast with parallel experiments using anti-PtdIns(4,5)P2 antibody, where only GST–PH-PLCδ1 was able to abolish staining, consistent with our earlier report [29]. Given the specific competition of PtdIns4P staining by exogenous PtdIns4P compared with PtdIns(4,5)P2 (Figure 1), it seems unlikely that this result reflects reactivity of anti-PtdIns4P antibody with plasma membrane PtdIns(4,5)P2. As argued above, it may simply reflect binding of high concentrations of GST–PH-PLCδ1 to plasma membrane PtdIns4P. However, given the enrichment of PtdIns(4,5)P2 at the plasma membrane, cross-reactivity was more of a concern than for Golgi staining, so we conceived further experiments to eliminate this possibility.

Bottom Line: PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2).Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus.In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK. gruh2@cam.ac.uk

ABSTRACT
PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2). Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

Show MeSH
Related in: MedlinePlus