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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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Specificity of the anti-PtdIns4P antibodyHeLa cells were fixed with 4% FA plus 0.2% GA and stained on ice using saponin permeabilization, as described in the Experimental section. Anti-PtdIns4P antibody was pre-absorbed with POPC liposomes containing 5% (mol/mol) of the indicated inositol lipid for 1 h at room temperature prior to applying to the cells on ice. Anti-PtdIns4P antibody staining is yellow, and DAPI-stained nuclei are in blue. Scale bar=20 μm. Images are single confocal optical sections.
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Figure 1: Specificity of the anti-PtdIns4P antibodyHeLa cells were fixed with 4% FA plus 0.2% GA and stained on ice using saponin permeabilization, as described in the Experimental section. Anti-PtdIns4P antibody was pre-absorbed with POPC liposomes containing 5% (mol/mol) of the indicated inositol lipid for 1 h at room temperature prior to applying to the cells on ice. Anti-PtdIns4P antibody staining is yellow, and DAPI-stained nuclei are in blue. Scale bar=20 μm. Images are single confocal optical sections.

Mentions: We have previously established conditions for staining of plasma membrane PtdIns(4,5)P2 using a specific monoclonal antibody [29]. Applying these conditions with a monoclonal anti-PtdIns4P antibody yielded plasma membrane staining in HeLa cells. The staining appeared specific, since it was abolished by pre-incubation of the antibody with liposomes containing 5 % (mol/mol) di-palmitoyl PtdIns4P, but not with any other inositol lipid (Figure 1). However, it was surprising to see such an exclusive plasma membrane staining, given the extensive evidence for a Golgi pool of PtdIns4P (described in the Introduction section).


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

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

Specificity of the anti-PtdIns4P antibodyHeLa cells were fixed with 4% FA plus 0.2% GA and stained on ice using saponin permeabilization, as described in the Experimental section. Anti-PtdIns4P antibody was pre-absorbed with POPC liposomes containing 5% (mol/mol) of the indicated inositol lipid for 1 h at room temperature prior to applying to the cells on ice. Anti-PtdIns4P antibody staining is yellow, and DAPI-stained nuclei are in blue. Scale bar=20 μm. Images are single confocal optical sections.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2722159&req=5

Figure 1: Specificity of the anti-PtdIns4P antibodyHeLa cells were fixed with 4% FA plus 0.2% GA and stained on ice using saponin permeabilization, as described in the Experimental section. Anti-PtdIns4P antibody was pre-absorbed with POPC liposomes containing 5% (mol/mol) of the indicated inositol lipid for 1 h at room temperature prior to applying to the cells on ice. Anti-PtdIns4P antibody staining is yellow, and DAPI-stained nuclei are in blue. Scale bar=20 μm. Images are single confocal optical sections.
Mentions: We have previously established conditions for staining of plasma membrane PtdIns(4,5)P2 using a specific monoclonal antibody [29]. Applying these conditions with a monoclonal anti-PtdIns4P antibody yielded plasma membrane staining in HeLa cells. The staining appeared specific, since it was abolished by pre-incubation of the antibody with liposomes containing 5 % (mol/mol) di-palmitoyl PtdIns4P, but not with any other inositol lipid (Figure 1). However, it was surprising to see such an exclusive plasma membrane staining, given the extensive evidence for a Golgi pool of PtdIns4P (described in the Introduction section).

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