Direct Visualization of Lipoprotein Mediated Cholesterol Transport at the Phospholipid Bilayer Interface

Abstract

It is still unclear how lipids are removed from the circulation and transferred from lipoproteins– the main carriers of cholesterol in the blood stream – to cells. Low density Lipoprotein (LDL)-uptake is facilitated mainly via receptor-mediated endocytosis in clathrin-coated pits but can also deliver its lipid cargo by selective transfer without degradation of the LDL particle. Whereas, High Density Lipoprotein (HDL) particles are captured from the blood stream by the corresponding receptor - the subsequent lipid-transfer process, however, is highly speculative. Here we used high-end techniques, namely combined atomic force (AFM) and single molecule fluorescence microscopy together with high speed AFM (HS-AFM) and fluorescence cross correlation spectroscopy (FCCS), to address the different transfer behaviour of lipids out of an LDL or HDL particle into a supported lipid membrane and their interaction kinetics. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of amphiphilic fluorescent probe lipids from lipoprotein particles to the lipid bilayer upon contact and their integration into the lipid bilayer was seen. FCCS experiments indicate that Cholesterol was freely mobile in the GUV membrane, additionally two color FCS revealed the absence of co-diffusion of the two species, indicating that cargo was transferred and separated from the particle. With the aid of HS-AFM we are able to achieve nanoscale spatial resolution together with real-time temporal resolution. Interestingly, on mica-supported DOPC membrane LDL in contrast to HDL was strongly interacting with the surface beneath by repetitive fusion and release. Taken together, in synthetic membranes, particle contact and incorporation into the lipid bilayer are sufficient requirements for cargo delivery. Whereby, the interaction kinetics and the integration of hydrophobic lipoprotein moieties in the core of the lipid bilayer strongly depends on the lipoprotein species.