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.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Cell Membrane/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Algorithms , Blood-Brain Barrier , Cell Line , Cell Separation , Cluster Analysis , Endothelial Cells/cytology , Flow Cytometry/methods , Genetic Vectors , Green Fluorescent Proteins/metabolism , Humans , Lentivirus/genetics , Microcirculation , Microscopy, Fluorescence/methodsABSTRACT
The aim of the study was to investigate the influence of diabetes mellitus type 1 on expression and function of the ATP-binding cassette transport proteins P-glycoprotein (Pgp, Abcb1) and breast cancer resistance protein (Bcrp, Abcg2) at the blood-brain barrier and the blood-cerebrospinal fluid barrier formed by the choroid plexus. In brain capillary endothelial cells forming the blood-brain barrier, Pgp and Bcrp are located in the luminal membrane while apical/sub-apical localization was described for Pgp in choroid plexus epithelial cells. Alterations in expression or function may lead to damages in barrier integrity and may cause brain defects after long term diabetes. Diabetes was induced by i.p.-streptozotocin injection 14days prior to performing experiments. RNA and protein expression were analyzed in choroid plexus and blood-brain barrier capillaries by RT-PCR and Western blot, respectively. Pgp and Bcrp expression was increased in blood-brain barrier capillaries; in choroid plexus, only Bcrp showed elevated gene expression. Protein expression was not altered. Functional analyses were carried out using confocal laser-scanning microscopy in intact isolated brain capillaries with the fluorescent Pgp substrate NBD-Cyclosporin A (NBD-CsA) and BODIPY® FL prazosin as substrate for Bcrp. Consistent with protein expression data, no changes in diabetic animals occurred, suggesting an unaltered function of Pgp and Bcrp.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP-Binding Cassette Transporters/genetics , Blood-Brain Barrier/metabolism , Choroid Plexus/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/biosynthesis , Animals , Blood-Brain Barrier/physiopathology , Choroid Plexus/physiopathology , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/physiopathology , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Up-Regulation/geneticsABSTRACT
Tendon cells are specialized cells of the insect epidermis that connect basally attached muscle tips to the cuticle on their apical surface via prominent arrays of microtubules. Tendon cells of Drosophila have become a useful genetic model system to address questions with relevance to cell and developmental biology. Here, we use light, confocal, and electron microscopy to present a refined model of the subcellular organization of tendon cells. We show that prominent arrays of F-actin exist in tendon cells that fully overlap with the microtubule arrays, and that type II myosin accumulates in the same area. The F-actin arrays in tendon cells seem to represent a new kind of actin structure, clearly distinct from stress fibers. They are highly resistant to F-actin-destabilizing drugs, to the application of myosin blockers, and to loss of integrin, Rho1, or mechanical force. They seem to represent an important architectural element of tendon cells, because they maintain a connection between apical and basal surfaces even when microtubule arrays of tendon cells are dysfunctional. Features reported here and elsewhere for tendon cells are reminiscent of the structural and molecular features of support cells in the inner ear of vertebrates, and they might have potential translational value.
