ABSTRACT
The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer's intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 µm microcarriers (MCs). Each MC functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of impedance measurement. We validated the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by fluorescence-assisted sorting from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Endothelial Cells/cytology , Genome-Wide Association Study/methods , High-Throughput Screening Assays/methods , RNA, Guide, Kinetoplastida/genetics , Adherens Junctions/physiology , Cell Line, Transformed , Clone Cells/metabolism , Collagen/metabolism , Endothelial Cells/metabolism , Fibronectins/metabolism , Flow Cytometry/methods , Fluorescent Dyes/analysis , Gelatin , Gene Library , Genome-Wide Association Study/instrumentation , High-Throughput Screening Assays/instrumentation , Humans , Male , Miniaturization , Permeability/drug effects , RNA Interference , Repressor Proteins/genetics , Signal Transduction/genetics , Thrombin/metabolism , Thrombin/pharmacology , Tight Junctions/physiology , Transcription, Genetic , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus.
