Drebrin preserves endothelial integrity by stabilizing nectin at adherens junctions

Abstract

SummaryRegulation of cell-cell contacts is essential for integrity of the vascular endothelium. Here, a critical role of the F-actin-binding protein drebrin in maintaining endothelial integrity is revealed under conditions mimicking vascular flow. Drebrin knockdown leads to weakening of cell-cell contacts, characterized by loss of nectin from adherens junctions and its subsequent lysosomal degradation. Immunoprecipitation, FRAP and mitochondrial re-targeting experiments show that nectin stabilization occurs through a chain of interactions: drebrin binding to F-actin, interaction of drebrin and afadin through their polyproline and PR1-2 regions, and recruitment of nectin through the PDZ region of afadin. Key elements are modules in drebrin that confer binding to afadin and F-actin. Evidence for this was obtained using constructs containing the PDZ region of afadin coupled to the F-actin-binding region of drebrin or to lifeact, which restore junctional nectin under knockdown of drebrin or of both drebrin and afadin. Drebrin, containing binding sites for both afadin and Factin, is thus uniquely equipped to stabilize nectin at endothelial junctions and to preserve endothelial integrity under vascular flow.

Requirement of the F‐actin‐binding activity of l‐afadin for enhancing the formation of adherens and tight junctions

“…Through the PDZ domain, both l‐afadin and s‐afadin bind to the cell adhesion molecule (CAM) nectin, which comprises a family with four members (nectin‐1, nectin‐2, nectin‐3, and nectin‐4) through their cytoplasmic tails (Takai, Ikeda, Ogita, & Rikitake, ; Takai, Miyoshi, Ikeda, & Ogita, ). In addition to nectin, many proteins, including Rap1 small G protein, αE‐catenin, p120 ctn , PLEKHA7, ZO‐1, ponsin, ADIP, LMO7, and drebrin, bind to l‐afadin through its respective binding domains (Asada et al., ; Boettner, Govek, Cross, & Van Aelst, ; Hoshino et al., ; Kurita, Yamada, Rikitsu, Ikeda, & Takai, ; Linnemann et al., ; Mandai, Rikitake, Shimono, & Takai, ; Mandai et al., ; Miyata et al., ; Ooshio et al., , ; Rehm, Panzer, van Vliet, Genot, & Linder, ; Tachibana et al., ; Takahashi et al., ).…”

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

“…5C). Further mutagenesis analysis with DBN1 mutants (31) revealed that the most N-terminal ADF-H domain within DBN1 is responsible for restriction of viruses and other cargo (SI Appendix, Fig. S10C).…”

Drebrin and Spermatogenesis

“…The drebrin-based protein complex is known to stabilize F-actin, modifies microfilament structure, or moderates the interactions of other actin-binding proteins with actin microfilaments. For instance, drebrin binds to afadin to connect F-actin to nectin-based integral membrane proteins (Rehm et al 2013). It competes with tropomyosin for F-actin binding and inhibits actin-binding and cross-linking activity of α-actinin (Ishikawa et al 1994).…”

“…Drebrin E, besides expresses similarly to Arp3 spatiotemporally during the epithelial cycle, also has high affinity to bind to Arp3, illustrating that drebrin E may recruit Arp3 to the ES to induce F-actin reorganization and to alter apical ES dynamics to facilitate spermiation. It has been reported that AJ integrity in endothelial cells is stabilized by drebrin since the knockdown of drebrin by RNAi in human umbilical vein endothelial cell (HUVEC), an endothelial cell line, leads to impairment of endothelial cell-cell junctions by causing mis-localization of afadin, thereby impeding the localization of nectin-2 and nectin-3 at the cell-cell interface (Rehm et al 2013). It is of interest to note that treatment of Sertoli cells with a functional TJ barrier in vitro with TNFα and TGFβ3 that downregulates drebrin E expression also increases structural interaction between drebrin E and Arp3 (Li et al 2011), illustrating an increase in the intrinsic branched actin polymerization activity.…”

“…In addition, spikar is shown to bind to the ADF-H domain (Yamazaki et al 2014). Drebrin also contains a proline-rich (PP) domain known to bind profilin (Mammoto et al 1998), afadin (Rehm et al 2013), and possibly Arp3 (Li et al 2011). There are also two homer-binding motifs near the C-terminus that interact with cupidin/homer2 (Shiraishi-Yamaguchi et al 2009) and also an SH2-binding motif.…”