Abstract
P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.
Highlights
The primary obstacle to the central nervous system (CNS) is the blood-brain barrier (BBB), which is formed by the brain capillary endothelial cells
The localization of Pgp-green fluorescent protein (GFP) fusion protein in hCMEC/D3 cells was determined after viral transduction with the construct
Note the scaling of the y-axis in Fig. 9, the peak is caused by several thousands of Pgp-GFP molecules in clustered formations. This is the first report to apply a Pgp-GFP construct on a human brain endothelial cell line to study the spatial distribution on a single molecule level with localization fluorescence microscopy (SPDM)
Summary
The primary obstacle to the central nervous system (CNS) is the blood-brain barrier (BBB), which is formed by the brain capillary endothelial cells These cells express multiple membrane-bound ATP-binding cassette (ABC) efflux transporters including Pglycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2), and several isoforms of multidrug resistanceassociated proteins (MRPs, ABCCs). They prevent the entry of xenobiotics and potentially toxic metabolites into the CNS and contribute to lowered drug accumulation within the brain [1,2,3,4]. The protein recognizes a remarkably broad diversity of molecules ranging from amphiphilic, to neutral or cationic structures [6] It is still not completely clear whether the transported substrates are released in the exoplasmic leaflet of a membrane or directly into the extracellular medium. As reviewed [9], Pgp is highly sensitive to its lipid environment, and the fluidity of the surrounding lipid rafts directly influences the activity of the export pump
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
