Selective Detection of Phospholipids in Model Cell Membranes by AFM Stylus Modified with Probe Protein

We detected sphingomyelin (SM), a ubiquitous kind of phospholipids, contained in model cell membranes, using AFM tips linked with lysenin, a probe protein that specifically binds SM [1]. The model membranes, containing phosphatidylcholines (DOPC or DPPC) and cholesterol as well as palmitoyl sphingomielin (PSM), were deposited on mica as double layers in an aqueous buffer solution. Smooth bilayers of pure PSM and PSM+cholesterol were discerned with tips without lysenin. The PSM+DOPC and PSM+DOPC+cholesterol areally were separated into two phases in the sub-micron scale. By lysenin-conjugated tips, on nearly 90% of the force-curve cycles, no attractive force was observed. In the rest 10% cases, we observed fairly constant adhesion forces ∼ 170 pN for the bilayers containing PSM. This force is less than one tenth of the force to remove a phospholipid molecule out of bilayer [2], and therefore recognized as the lysenin single-molecular bonding force with PSM. Lysenin tips were active for averagely 1000 approaches to PSM containing membranes. Some tips were never active. We plotted the distribution of adhesive force over the two phases seen on the PSM+DOPC(+cholesterol) bilayers. In one of two phases, the adhesive forces ∼ 170 pN were observed, and in the other phase, adhesive forces were not measured. The phase condensation of PSM was apparently demonstrated by the distribution of force curves. The bilayer lipid phase containing PSM and cholesterol corresponds to the “raft” structure on the real cell membranes, anticipated to be instrumental in cellular signaling as well as virus infection. Our present technique will be applicable for direct observation of the raft on real live cell membranes.[1] T. Wang et al., Biochemistry 51 (2012) 74.[2] R. M. Sullan et al., Langmuir25 (2009) 7471.