Phase evolution in cholesterol/DPPC monolayers: atomic force microscopy and near field scanning optical microscopy studies

Abstract

SummaryA combination of atomic force microscopy (AFM) and near field scanning optical microscopy has been used to study domain formation in dipalmitoylphosphatidylcholine (DPPC)/ cholesterol monolayers with cholesterol concentrations ranging from 0 to 50%. The results show a clear evolution from a mixture of liquid expanded and liquid condensed phases for cholesterol concentrations < 10% to a mixture of liquid expanded and two cholesterol-containing phases at intermediate concentrations, and finally to a single homogeneous liquid ordered phase for 33% cholesterol. Mixtures of the various phases are clearly identified by height differences in AFM and in some cases by fluorescence imaging for samples containing 0.5% BODIPY dye, which localizes preferentially in the more fluid phase. Note that fluorescence imaging, at least with the dye used here, is unable to distinguish between the cholesterolrich and cholesterol-poor phases detected at intermediate cholesterol concentrations. The combination of fluorescence and AFM imaging provides a more complete picture of the phase evolution for cholesterol/DPPC monolayers than could be obtained by either technique alone, and presents substantial advantages over conventional fluorescence microscopy in that submicrometre-sized domains can be readily detected.

Surface rheology of monolayers of phospholipids and cholesterol measured with axisymmetric drop shape analysis

“…The isotherm of cholesterol shows a single phase with a collapse pressure of approximately 45 mN m −1 , at which point the molecular area is ∼39Å 2 [15,46,49]. It remains in the solid condensed phase up to the point of monolayer collapse [46].…”

The interfacial bioscience grand challenge.

“…As shown in Figure 2.5b, the internal ion b(19-32) does not appear modified in any of the MS/MS spectra, indicating that Lys 27 and Lys 29 are among the least reactive residues in the protein. The internal b (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) ion, in contrast, is singly acetylated in the MS/MS spectrum of the doubly acetylated ubiquitin (Figure 2.5c). b (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) contains Lys 27, Lys 29, and Lys 33, therefore Lys 33 must be the reactive residue, since Lys 27 and Lys 29 were shown to be unmodified in the MS/MS spectrum of doubly acetylated ubiquitin in Figure 2.5b.…”

“…For each sample, a consistent and reproducible cleavage pattern was observed following treatment with CNBr, facilitating the mass spectral analysis and assignment of the rhodopsin peptide fragments (Figure 1.2a). Several groups have reported the use of C4, C8 or C18 columns for the RP-HPLC separation of digested peptides from integral membrane proteins including rhodopsin 26,29 . We achieved optimal separation using a polystyrene/divinylbenzene column (PLRP-S, Michrom Bioresources) for the detection of hydrophobic and cross-linked rhodopsin fragments following CNBr cleavage.…”

“…Integral membrane proteins pose a significant analytical challenge due to their amphiphillic nature and their tendency to aggregate and adhere to glass, plastic, and commonly used HPLC stationary phases. However, several groups have recently made progress in the preparation of integral membrane proteins, including rhodopsin, for mass spectrometry [26][27][28][29][30] . In our work, selected methods were optimized in order to maximize the recovery of cross-linked rhodopsin for subsequent LC-MS and tandem MS analysis.…”

“…Thus many studies of lipid domains in model systems have focused on phase separation within multicomponent lipid mixtures, on the basis of head group interactions and/or acyl chain structure, and the subsequent partitioning of protein-binding glycosphingolipids between the phases. Domain structure in bilayer and monolayer assemblies has been characterized by numerous fluorescence microscopy methods including far-field 14,15,17,26 and near-field imaging [27][28][29] , fluorescence recovery after photobleaching 10,13 , and fluorescence quenching 16,18 . Indeed, these techniques have been useful for domain studies on cellular membranes as well 19,[30][31][32][33][34] .…”