The human endothelium forms a permeable barrier between the blood stream and the surrounding tissue, strictly regulating the passage of immune cells and metabolites. For its correct function, regulation of cell-cell contact dynamics between endothelial cells is essential. In this context, we focus particularly on the adherens junctions system, which is predominantly composed of intercellular adhesion proteins such as VE-cadherin and nectin, as well as their associated proteins.
Drebrin, an F-actin binding protein has been included in the growing list of “junction associated” proteins, but its potential role in adherens junction dynamics has been unclear so far. We could show that knockdown of drebrin leads to functional impairments of endothelial monolayers, shown by rupturing of HUVEC monolayers cultured under constant unidirectional flow conditions, which mimic blood flow in vessels.
The observed weakening of cell-cell contacts is characterized by a specific and complete loss of nectin from adherens junctions, due to its endocytosis and subsequent degradation in lysosomes.
Performing co-immunoprecipitation and GST-pulldown experiments, we could show that drebrin does not interact with nectin-2 directly, but with its binding partner, afadin. This was further confirmed by mitochondrial retargeting experiments, where drebrin’s afadin-binding polyproline region fused to a mitochondrial targeting signal is sufficient to relocalize afadin towards the outer membrane of mitochondria.
Furthermore, we could demonstrate that drebrin’s role in maintaining junctional integrity is to link the nectin/afadin system to the cortical F-actin network. Evidence is provided by expressing minimal rescue constructs containing exclusively afadin´s PDZ region (binding nectin) coupled to drebrin´s F-actin binding region, which restores junctional nectin under knockdown of both drebrin and afadin.
These results contribute to our current understanding of how junctions are regulated in the endothelium under vascular flow. In particular, the newly identified interaction between drebrin and afadin is shown to be crucial for nectin-based junctional integrity. They also indicate that, contrary to previous assumptions, nectins are not only important for formation of cell-cell junctions but also for their upkeep. Current work concerns the dynamic regulation of the F-actin-drebrin-afadin-nectin chain in maintaining endothelial integrity.