Abstract
Phosphoinositides have been shown to control membrane trafficking events by targeting proteins to specific cellular sites, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. To shed light on the processes that lead to the formation of phosphoinositide-enriched microdomains, mixed monolayers of phosphatidylcholine and dioleoyl-phosphatidylinositol (DOPtdIns) or dioleoyl-phosphatidylinositol-bisphosphate [DOPtdIns(4,5)P(2)] were investigated by isothermal area/pressure measurements, Brewster angle microscopy, and grazing incidence X-ray diffraction. The results are consistent with a charge-dependent formation of phosphatidylinositol-containing tightly packed phases. DOPtdIns is capable of mixing partially with condensed 1,2-distearoyl-phosphatidylcholine (DSPC) and of forming mixed crystals that differ significantly from those formed by pure DSPC. DOPtdIns(4,5)P(2) in mixtures with DSPC is, to a much larger extent, phase separated. The observed phase separation of the highly charged DOPtdIns(4,5)P(2) is presumably water stabilized by electrostatic interactions and hydrogen bonding. In biological systems, an enzymatic phosphorylation of phosphatidylinositol in mixed domains may cause their insolubility in ordered phosphatidylcholine areas and lead to a cooperative reorganization of the host lipid membrane. This strong cooperative effect underlines the important role of PtdIns(4,5)P(2) in signal transduction processes and suggests that the ability of phosphoinositides to induce or reduce long-range interactions in phospholipid mixtures is crucial.
Highlights
Phosphoinositides have been shown to control membrane trafficking events by targeting proteins to specific cellular sites, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization
If one compares the lattice parameters of the pure DSPC and the DSPC/DOPtdIns(4,5)P2 mixture, slightly smaller tilt angles and unit cell dimensions can be found for the mixed monolayer
Based on the presented results, DSPC/DOPtdIns(4,5)P2 mixtures form condensed phase domains containing mainly DSPC and small amounts of DOPtdIns(4,5)P2 surrounded by a fluid phase composed of predominantly DOPtdIns(4,5)P2, perhaps containing a very small amount of DSPC
Summary
The morphology of the monolayer was visualized with a Brewster angle microscope (BAM1) (Nanofilm Technology; Göttingen, Germany) mounted on a Langmuir film balance (R & K). A helium-neon laser (10 mW) produces light with a wavelength of 632.8 nm. The p-polarized light is directed to the water surface under the Brewster angle. The diameter of the beam is 0.68 mm. The trough is mounted on a X-Y translation table (Märzhäuser; Wetzlar, Germany), which is placed on an anti-vibrational table (JAS; Affoltern, Switzerland) (13, 14). Image processing software was used to correct the Brewster angle microscopy (BAM) images for the distortion due to the observation at the Brewster angle. BAM is sensitive to changes in layer thickness and orientation of the aliphatic chains. The resolution of the BAM1 is about 4 mm. The images shown here are 500·500 mm[2] in size (scale bars indicated)
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