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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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Effect of a 5-phosphatase on plasma membrane PtdIns4P/PtdIns(4,5)P2 labellingCOS-7 cells were transfected with GFP, GFP-Pharbin or the putatively inactive mutant of the latter, D559A, for 18 h prior to fixation and staining as described in Figure 7. (a) Representative images of COS-7 cells transfected with each construct. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. (b) Quantification of staining (n=93 cells). See the Experimental section for details. *P<0.05, **P<0.01.
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Figure 9: Effect of a 5-phosphatase on plasma membrane PtdIns4P/PtdIns(4,5)P2 labellingCOS-7 cells were transfected with GFP, GFP-Pharbin or the putatively inactive mutant of the latter, D559A, for 18 h prior to fixation and staining as described in Figure 7. (a) Representative images of COS-7 cells transfected with each construct. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. (b) Quantification of staining (n=93 cells). See the Experimental section for details. *P<0.05, **P<0.01.

Mentions: Expression of an inositol 5-phosphatase activity would be expected to decrease PtdIns(4,5)P2 levels in the plasma membrane without depletion of PtdIns4P. To this end, we transfected COS-7 cells with GFP-tagged pharbin, an enzyme with PtdIns(4,5)P2 5-phosphatase activity [52]. Cells were then fixed and co-stained with an anti-PtdIns4P antibody and anti-GST antibody to stain for GST–PH-PLCδ1 and follow PtdIns(4,5)P2 [31]. Over-expressed pharbin was localized throughout the cytosol and was particularly enriched on the Golgi, as reported previously in [52,53]. Despite its preference for PtdIns(3,4,5)P3 as a substrate [53], the over-expressed enzyme caused reduction in the intensity of staining against PtdIns(4,5)P2 compared with neighbouring cells without visible GFP expression. This effect was not seen in GFP-expressing cells or in cells expressing a mutant pharbin, with the putative catalytic aspartate-559 residue mutated to alanine (Figure 9a). Quantification of fluorescence images showed that pharbin caused an approx. 50% decrease in staining for PtdIns(4,5)P2 (Figure 9b). Importantly, no reduction in staining for PtdIns4P was detected (Figure 9b), and in fact the quantification revealed a slight increase in PtdIns4P staining. This is probably not due to catalytic production of PtdIns4P by pharbin as it is also seen with the inactive mutant (Figure 9b).


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

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

Effect of a 5-phosphatase on plasma membrane PtdIns4P/PtdIns(4,5)P2 labellingCOS-7 cells were transfected with GFP, GFP-Pharbin or the putatively inactive mutant of the latter, D559A, for 18 h prior to fixation and staining as described in Figure 7. (a) Representative images of COS-7 cells transfected with each construct. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. (b) Quantification of staining (n=93 cells). See the Experimental section for details. *P<0.05, **P<0.01.
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Related In: Results  -  Collection

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Figure 9: Effect of a 5-phosphatase on plasma membrane PtdIns4P/PtdIns(4,5)P2 labellingCOS-7 cells were transfected with GFP, GFP-Pharbin or the putatively inactive mutant of the latter, D559A, for 18 h prior to fixation and staining as described in Figure 7. (a) Representative images of COS-7 cells transfected with each construct. Images are maximum intensity projections of confocal sections spanning the entire depth of the cells. Scale bar=20 μm. (b) Quantification of staining (n=93 cells). See the Experimental section for details. *P<0.05, **P<0.01.
Mentions: Expression of an inositol 5-phosphatase activity would be expected to decrease PtdIns(4,5)P2 levels in the plasma membrane without depletion of PtdIns4P. To this end, we transfected COS-7 cells with GFP-tagged pharbin, an enzyme with PtdIns(4,5)P2 5-phosphatase activity [52]. Cells were then fixed and co-stained with an anti-PtdIns4P antibody and anti-GST antibody to stain for GST–PH-PLCδ1 and follow PtdIns(4,5)P2 [31]. Over-expressed pharbin was localized throughout the cytosol and was particularly enriched on the Golgi, as reported previously in [52,53]. Despite its preference for PtdIns(3,4,5)P3 as a substrate [53], the over-expressed enzyme caused reduction in the intensity of staining against PtdIns(4,5)P2 compared with neighbouring cells without visible GFP expression. This effect was not seen in GFP-expressing cells or in cells expressing a mutant pharbin, with the putative catalytic aspartate-559 residue mutated to alanine (Figure 9a). Quantification of fluorescence images showed that pharbin caused an approx. 50% decrease in staining for PtdIns(4,5)P2 (Figure 9b). Importantly, no reduction in staining for PtdIns4P was detected (Figure 9b), and in fact the quantification revealed a slight increase in PtdIns4P staining. This is probably not due to catalytic production of PtdIns4P by pharbin as it is also seen with the inactive mutant (Figure 9b).

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