Super Resolution imaging enables Nano-scale molecular imaging of endothelial transcytosis

Blood Brain Barrier (BBB) endothelial cells maintain low rate of vesicular transport (transcytosis). This cell biological property contributes to high degree of barrier selectivity. Inhibition of BBB transcytosis is acquired during embryonic development, tightly controlled in adults and deregulated in pathological conditions, especially in Alzheimer’s disease and in stroke-related BBB dysfunction.

Here we describe a super-resolution imaging (STORM) approach enabling Nano-scale molecular imaging of endothelial transcytosis. We combine imaging of structural and regulatory proteins (e.g CAV1 and MFSD2A) together with various cargo molecules, all at the single molecule and Nano-scale resolution. Our study includes imaging ofendothelial cellsin vitro, imaging of lung tissue as a model for non-barrier endothelial cells and imaging of BBB endothelial cells (in brain tissue) in different developmental stages of barrier formation.

We demonstrate the ability to quantify the amount and localization of CAV1 and MFSD2a proteins. Based on our preliminary data we challenge the traditional view of CAV1 as a vesicular ‘decorating’ protein and find that it has a focal organization associated with cargo. We also use this imaging approach together with gain of expression to examine MFSD2A effects on transcytosis inhibition.

Super-resolution imaging provides a novel point of view on protein interactions at the BBB leading to better understanding the molecular mechanisms of BBB selectivity. Shading light on mechanisms controlling BBB transcytosis may assist restoring suppression of pathological transcytosis and open new directions to manipulate the BBB for CNS drug delivery.