Claudin5 – not what you would expect; new BBB tight junction organization and function revealed through super-resolution imaging

Unique tight junctions of BBB endothelial cells are considered a fundamental hallmark of barrier function. Major efforts have been invested in tight junctions research and in particular in the prototypical junctional protein Claudin5 (CLD5). CLD5 deficient mice display a size selective barrier defect. Changes in CLD5 levels and organization were found in many pathological conditions.
Here we use stochastic optical reconstruction microscopy (STORM) for super-resolution imaging of endothelial tight junctions. STORM combines high resolution together with the versatility of fluorescence microscopy, enabling Nano-scale molecular imaging of tight junction proteins together with various tracer molecules. Focusing on CLD5 during the developmental formation and maturation of the BBB revealed unexpected findings regarding tight junction organization and function:
First, we show that CLD5 is organized in clusters rather then in strands. Second, our approach enables direct visualization of tracer leakage through immature BBB tight junctions at very early stages of embryonic development (E12.5). Third, most surprisingly we find an inverse correlation between CLD5 abundance and BBB permeability along development, suggesting that less CLD5 proteins results in tighter barrier properties. Finally, changes in BBB selectivity in CLD5 null mice were speculated to be the result of removing CLD5 physical obstruction to small molecules. We find irregular changes in the organization of tight junction proteins in these mice, which might provide alternative explanation to changes in barrier selectivity and warrant further investigation. Our study offers a novel approach that will enhance our fundamental understanding of the structure and function of BBB tight junctions.