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Drebrin preserves endothelial integrity by stabilizing nectin at adherens junctions
Drebrin reguliert die Integrität des Endothels durch Stabilisation von Nectin an Adherenzkontakten
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Freie Schlagwörter (Deutsch):
Drebrin Nectin Afadin Endothel Adherenzkontakt
Freie Schlagwörter (Englisch):
drebrin nectin afadin endothelium adherens-junction
Linder, Stefan (Prof. Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
The human endothelium forms a permeable barrier between the blood stream and surrounding tissue, strictly governing the passage of immune cells and metabolites. Regulation of cell-cell contact dynamics between endothelial cells is essential for the maintenance of its function and the vascular integrity. Besides other junctional systems, the accurate adhesion of cells is mainly dependent 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, a protein mainly expressed in neurons, has been included in the growing list of “junction associated” proteins, but its exact role in adherens junction dynamics has so far been unclear.
In this study, we show that knockdown of drebrin leads to functional impairments of endothelial monolayers, as demonstrated by a decrease of transendothelial electrical resistance (TER) and rupturing of HUVEC monolayers cultured under constant unidirectional flow conditions. The observed weakening of cell-cell contacts upon drebrin depletion is characterized by a specific and complete loss of nectin from adherens junctions, due to its endocytosis and subsequent degradation in lysosomes. The importance of drebrin for nectin’s presence at junctions is underlined by rescue experiments, where transient re expression of siRNA-insensitive drebrin stabilizes nectin at the junctional area. To support the fact that the phenotype is indeed due to loss of nectin, we established a knockdown of all endothelial nectin isoforms, which resulted in an even more pronounced phenotype than drebrin knockdown.
Conducting co-immunoprecipitation experiments, we could show that drebrin does not interact with nectin directly but with its most prominent intracellular binding partner, afadin. Direct binding of drebrin and afadin is mediated through their polyproline and PR1-2 regions as shown by GST pulldown experiments using bacterially expressed domain constructs of both proteins. Concomitantly, confocal microscopy studies revealed strong binding between the two proteins on a subcellular level: drebrin’s polyproline region fused to a mitochondrial targeting signal is sufficient to relocalize afadin towards the outer membrane of mitochondria. Due to its association with nectin, afadin is also affected by drebrin knockdown – its mobility at the junctional area is enhanced, as shown by FRAP experiments, even though it still localizes to junctions, probably through binding other proteins, such as ZO-1 or α-catenin.
Furthermore, we could demonstrate that drebrin maintains junctional integrity through its ability to link the nectin/afadin system to the cortical F-actin network. Being equipped with an F-actin binding module (CC-region) and the afadin-binding polyproline region, it anchors afadin to F-actin. Simultaneously, afadin binds to nectin through its PDZ region, resulting in a chain of protein interactions: F-actin-drebrin-afadin-nectin, which indirectly stabilizes nectin at the F-actin network (Figure 46A). Evidence, verifying that linkage of nectin to F actin is essential for monolayer integrity is provided by rescue of junctional nectin, under knockdown of both drebrin and afadin, through overexpression of minimal constructs containing exclusively afadin´s PDZ region coupled to drebrin´s F-actin binding region, or lifeact (Figure 46C). Drebrin, containing binding sites for both afadin and F-actin, is thus uniquely equipped to stabilize nectin at endothelial junctions, thereby preserving endothelial integrity. Altogether, these results contribute to the current understanding of how junctions are regulated in the endothelium under vascular flow, especially elucidating the significance of nectins. In particular, the newly identified interaction between drebrin and afadin, which establishes the necessary linkage of nectins to cortical F-actin, is shown to be crucial for junctional integrity.