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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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Related in: MedlinePlus

Effect of fixation, detergent and temperature on the retention of various organelle-specific lipid stainsCells were fixed with the indicated concentration of aldehydes, then stained at the designated temperature after permeabilization with digitonin or saponin. See the Experimental section for details. Agents were used that specifically label the (a) Golgi (NBD-ceramide), (b) plasma membrane (anti-PtdIns(4,5)P2 antibody), (c) endosomes (anti-GST against GST–2×FYVE-Hrs bound to PtdIns3P) or (d) endoplasmic reticulum (DiOC6). DAPI-stained nuclei are shown in blue. Scale bars=20 μm.
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Figure 2: Effect of fixation, detergent and temperature on the retention of various organelle-specific lipid stainsCells were fixed with the indicated concentration of aldehydes, then stained at the designated temperature after permeabilization with digitonin or saponin. See the Experimental section for details. Agents were used that specifically label the (a) Golgi (NBD-ceramide), (b) plasma membrane (anti-PtdIns(4,5)P2 antibody), (c) endosomes (anti-GST against GST–2×FYVE-Hrs bound to PtdIns3P) or (d) endoplasmic reticulum (DiOC6). DAPI-stained nuclei are shown in blue. Scale bars=20 μm.

Mentions: Figure 2 shows the results from such a study, where strength of fixation was varied (by increasing FA concentration and adding GA), staining at room temperature was compared to staining on ice, and the permeabilizing detergent digitonin was compared with saponin. Using NBD-C6-ceramide as a post-fixation marker for Golgi membranes [30], it is clear that staining is abolished by saponin, whereas it persists when digitonin is employed (Figure 2a). Fixation and temperature conditions did not seem to drastically affect the staining in the presence of digitonin, although distinct Golgi staining was clearer when GA was omitted (probably because there is reduced autofluorescence in the cells).


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 fixation, detergent and temperature on the retention of various organelle-specific lipid stainsCells were fixed with the indicated concentration of aldehydes, then stained at the designated temperature after permeabilization with digitonin or saponin. See the Experimental section for details. Agents were used that specifically label the (a) Golgi (NBD-ceramide), (b) plasma membrane (anti-PtdIns(4,5)P2 antibody), (c) endosomes (anti-GST against GST–2×FYVE-Hrs bound to PtdIns3P) or (d) endoplasmic reticulum (DiOC6). DAPI-stained nuclei are shown in blue. Scale bars=20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of fixation, detergent and temperature on the retention of various organelle-specific lipid stainsCells were fixed with the indicated concentration of aldehydes, then stained at the designated temperature after permeabilization with digitonin or saponin. See the Experimental section for details. Agents were used that specifically label the (a) Golgi (NBD-ceramide), (b) plasma membrane (anti-PtdIns(4,5)P2 antibody), (c) endosomes (anti-GST against GST–2×FYVE-Hrs bound to PtdIns3P) or (d) endoplasmic reticulum (DiOC6). DAPI-stained nuclei are shown in blue. Scale bars=20 μm.
Mentions: Figure 2 shows the results from such a study, where strength of fixation was varied (by increasing FA concentration and adding GA), staining at room temperature was compared to staining on ice, and the permeabilizing detergent digitonin was compared with saponin. Using NBD-C6-ceramide as a post-fixation marker for Golgi membranes [30], it is clear that staining is abolished by saponin, whereas it persists when digitonin is employed (Figure 2a). Fixation and temperature conditions did not seem to drastically affect the staining in the presence of digitonin, although distinct Golgi staining was clearer when GA was omitted (probably because there is reduced autofluorescence in the cells).

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