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A Distinct Pool of Phosphatidylinositol 4,5-Bisphosphate in Caveolae Revealed by a Nanoscale Labeling Technique
Akikazu Fujita, Jinglei Cheng, Kumi Tauchi-Sato, Tadaomi Takenawa, Toyoshi Fujimoto and Pietro V. De Camilli
Proceedings of the National Academy of Sciences of the United States of America
Vol. 106, No. 23 (Jun. 9, 2009), pp. 9256-9261
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/40483202
Page Count: 6
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Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [Pl(4,5) P₂] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of Pl(4,5) P₂ at the nanoscale, we developed an electron microscopic technique that probes the f reeze-f ractured membrane preparation by the pleckstrin homology domain of phospholipase C-δ1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the Pl(4,5) P₂ distribution quantitatively. By using this method, we found that Pl(4,5) P₂ is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. Pl(4,5) P₂ was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the Pl(4,5) P₂ level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the Pl(4,5) P₂ level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the Pl(4,5) P₂ level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct Pl(4,5) P₂ pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling.
Proceedings of the National Academy of Sciences of the United States of America © 2009 National Academy of Sciences