Keywords

1 Drebrin in Monolayer-Forming Cells

Cell monolayers fulfill critical roles in multicellular organisms (Harris et al. 2012). Monolayers act by separating different tissues or compartments from each other, forming semipermeable barriers that can withstand mechanical forces. This is especially important in the case of epithelial and endothelial cells. The endothelium , in particular, serves as a semi-selective barrier between the bloodstream and the surrounding tissue, controlling the passage of leukocytes and solutes. Depending on the location of the endothelium in the vascular tree, the respective degree of permeability is quite variable. In this respect, specific and regulated anchorage between individual cells within monolayers is of major importance. Accordingly, dysregulation of cell–cell junctions compromises endothelial integrity and can thus lead to pathological scenarios such as thrombogenesis (Vestweber 2007; Muller 2003). In recent years, it has become increasingly clear that the various systems of intercellular cell–cell junctions (see Sect. 21.1.1) depend on the filamentous (F)-actin cytoskeleton for their stabilization. The regulation of F-actin dynamics is thus of major importance for the coherence and adaptability of the endothelium (Lampugnani 2010). This implies critical roles for F-actin-associated proteins in the maintenance of vascular integrity.

In this context, Peitsch et al. described drebrin E in a variety of cell types and tissues, also including epithelial and endothelial cells (Peitsch et al. 1999). Interestingly, drebrin levels are strongly increased in epidermal skin tumors, where it localizes to junctional areas. Of note, even at moderate levels of expression, such as in cultured keratinocytes, drebrin is enriched at adherens junctions (Peitsch et al. 2005). In line with these findings, confluent endothelial monolayers show a pronounced expression of drebrin E, with the majority being enriched at the cell cortex, where it associates with F-actin filaments, and preferentially near adherens junctions (Peitsch et al. 1999).

In our study, we were able to confirm the localization of drebrin to endothelial cell–cell junctions in human umbilical vein endothelial cells (HUVEC ) and focused in the following on the as yet unclear function of drebrin in endothelial junction regulation (Rehm 2013). In particular, we studied the dynamic behavior of endothelial monolayers that were cultivated under constant flow conditions of 15 dyne/cm2, thus mimicking flow conditions in medium-sized vessels (Paz et al. 2012). As expected, both control and drebrin-depleted cells aligned themselves along the direction of flow. However, in contrast to control monolayers that retained their integrity, monolayers of drebrin-depleted cells showed ruptures after 3 days, pointing to a role of drebrin in the maintenance of endothelial integrity especially under shear stress, probably through regulation of cell–cell junctions (Rehm et al. 2013) (Fig. 21.1).

Fig. 21.1
figure 1

Monolayers of HUVEC depleted for drebrin show ruptures when cultured under constant flow. Images of HUVEC monolayers, treated with drebrin-specific (a, c) or control siRNA (b, d) and seeded in Microslides. Cells were submitted to constant fluid shear stress for 1–3 days. The enlarged images (ai–di) show boxed regions of (a–d). Arrows indicate the direction of flow. Note the rupture of drebrin knockdown monolayers at day 3. Bars, 10 μm in (ai–di), 100 μm in (a–d) (Rehm et al. 2013)

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1.1 Different Sets of Cell–Cell Junctions Are Involved in Maintaining Monolayer Integrity

Within monolayers, cells are connected by a number of different junctional systems, which regulate endothelial barrier function, tissue integrity, as well as cell–cell communication (Wallez and Huber 2008; Lampugnani and Dejana 1997). These junctional systems, including tight junctions, adherens junctions, gap junctions, and desmosomes, have been studied especially in epithelial cells. In these cells, the different junctions follow a well-defined distribution along the intercellular cleft, with tight junctions being the most apical component, followed by adherens junctions and desmosomes. However, in endothelial cells, the junction types are less well organized and do not exhibit such a clear progression along the contact zone (Engelhardt and Wolburg 2004). Moreover, desmosomes are missing in endothelial cells, and instead, typical endothelial proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1) and intercellular adhesion molecule-2 (ICAM-2 ) are found, which contribute to cell–cell adhesion and also to angiogenesis or leukocyte extravasation (Muller et al. 1993; DeLisser et al. 1994; Lyck et al. 2003). Still, junctions of the epithelium and endothelium are highly homologous, which often allows a careful transfer of observations made in epithelial cell models also to endothelial cells.

1.1.1 Tight Junctions

In epithelial cells, tight junctions are found at the most apical position (Dejana et al. 2009). Their main function is the formation of an impermeable barrier for soluble molecules. In endothelial cells, the necessity for permeability control is not as absolute and can vary, depending on the location within the vascular tree (Bazzoni and Dejana 2004). The most prominent components of tight junctions are occludins, claudins, and junctional adhesion molecules (JAMs), with the latter also occurring in cells that do not form junctions (Bazzoni 2003). Occludins have four membrane-spanning regions, with their N- and C-termini both being intracellular. The 15 claudins identified so far show a similar architecture, with claudin-5 being the endothelial-specific member (Morita et al. 1999). Occludin and claudin can bind to different zonula occludentes (ZO) proteins (Tsukita et al. 1999; Tsukita and Furuse 1999), which primarily serve as linkers to the F-actin cytoskeleton and to other proteins (Stevenson et al. 1986).

1.1.2 Gap Junctions

In addition to the junctional complexes that serve as anchoring structures, gap junctions mediate communication between neighboring cells (Simon and Goodenough 1998). They constitute clusters of few to hundreds of intercellular channels, which are permeable for ions and metabolites, excluding molecules that exceed 1 kDa in size (Alexander and Goldberg 2003). Each channel is formed by transmembrane proteins that belong to the connexin (Cx) family, which consists of 20 members in humans, with Cx43, Cx40, and Cx37 being expressed in the endothelium. Connexins assemble into hexameric clusters, forming a hemichannel (connexon) in the plasma membrane, which aligns with another connexon of an adjacent cell, thus forming a pore that connects their cytoplasms (Bazzoni and Dejana 2004). This way, adjoined cells can share second messengers or metabolites and can give coordinated responses to extracellular stimuli. Of note, Butkevich et al. found drebrin at gap junctions of green monkey kidney epithelial cells. Moreover, siRNA-mediated depletion of drebrin led to impaired cell–cell coupling and internalization of connexin-43, thus destabilizing gap junctions (Butkevich et al. 2004).

1.1.3 Adherens Junctions

Adherens junctions (AJ) are molecular assemblies of proteins, which provide intercellular adhesion. Among the most prominent components are cadherins , with VE (vascular endothelial)-cadherin being the only one of over 350 cadherins that is expressed exclusively in endothelial cells (Hulpiau and van Roy 2011). VE-cadherin is part of the subfamily of classical cadherins, which all share six conserved extracellular cadherin domains responsible for calcium-dependent dimerization (Boggon et al. 2002). Besides their main function of mediating adhesion, cadherins play a role in intracellular signaling, as their cytoplasmic tail region interacts with a variety of proteins. Of major importance is p120-catenin, which binds to the juxtamembrane region of cadherins, preventing their clathrin-related endocytosis through stabilization at the membrane (Davis et al. 2003; Chiasson et al. 2009). Upon the release of p120-catenin, it is able to translocate into the nucleus to regulate transcription, displaying a dual role in the cell that is common for many junction-associated proteins (Cavallaro and Dejana 2011; Yap et al. 1997). Another prominent binding partner of cadherins is β-catenin , which was thought to link cadherins to F-actin through α-catenin (Gates and Peifer 2005). However, this model has been challenged, for example, through FRAP experiments showing that F-actin has a more dynamic behavior than α-catenin, which excludes the possibility of a stable complex (Yamada et al. 2005; Drees et al. 2005). Accordingly, Drees et al. showed that α-catenin either binds to β-catenin/cadherin or to F-actin but not to both at the same time (Drees et al. 2005; Weis and Nelson 2006).

Nectins constitute a further group of AJ-localized intercellular adhesion molecules (Lopez et al. 1995). They work independently of Ca2+ and consist of a cytoplasmic tail region, a single transmembrane region and three extracellular immunoglobulin-like loops involved in cis- and trans-oligomerization. Comparable to cadherins, nectins initially form lateral homo (or hetero-)-cis-dimers, followed by trans-interactions with a dimer of the adjacent cell (Satoh-Horikawa et al. 2000; Reymond et al. 2001). Currently, four members of the nectin family have been identified (nectin-1, 2, 3, and 4), with several respective splice variants (Satoh-Horikawa et al. 2000; Reymond et al. 2001). The major nectin isoforms in endothelial cells, including HUVEC, are nectin-2 and nectin-3 (Lopez et al. 1998). The cytoplasmic tail of nectins contains the motif E/A-X-Y-V, which binds the Postsynaptic density protein-95/Drosophila disc large tumor suppressor/Zonula occludens-1 protein (PDZ) domain of their typical binding partner afadin (Mandai et al. 1997).

Afadin (or l-Afadin) is a multivalent adaptor protein featuring a variety of functional domains, two Ras-associated domains (RA), one dilute (DIL) domain, a fork head-associated (FHA) domain, a PDZ domain, three proline-rich regions (PR), and an F-actin-binding C-terminus, allowing interactions with a variety of other proteins (Takahashi et al. 1999; Takai et al. 2008) (Fig. 21.2). Afadin is thought to serve as a connector between the two main adhesion systems, nectins, and cadherins, by binding nectin and several of the cadherin-associated proteins. Among others, it can bind to α-catenin , which is mainly localized at cadherin-based junctions and to ponsin, which then binds vinculin (Pokutta et al. 2002; Mandai et al. 1999).

Fig. 21.2
figure 2

Drebrin and afadin interact through their polyproline and PR1-2 regions. Upper scheme: Drebrin domain structure, including ADF-homology region (aa 8–134), coiled coil region involved in homodimerization and F-actin binding (Peitsch et al. 2001) (CC, aa 176–256), minimal actin-remodeling region (Hayashi et al. 1999) (MAR, aa 233–317), and polyproline region (PP, aa 364–417). Lower scheme: Afadin domain structure, including RA regions involved in Rap1 binding (aa 30–347), FHA region (aa 371–487), DIL region (aa 647–892), PDZ region involved in nectin binding (aa 1016–1100), PR1-2 region containing two polyproline stretches (aa 1219–1399), and FAB region involved in F-actin binding (aa 1691–1829), containing a third proline-rich stretch (PR3). Middle box: Illustration of direct interaction of drebrin’s PP and afadin’s PR1–2 regions. Western blots of pulldown assay using drebrin–PP fused to MBP (MBP-drebrin-PP) or MBP as control immobilized on amylose resin beads, incubated with afadin-PR1-2 fused to GST (GST-afadin-PR1-2). ± indicates presence of respective components in experiments. Western blots developed with indicated antibodies (Rehm 2013; Rehm et al. 2013)

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2 Junctional Integrity Depends on the F-actin Cytoskeleton

The cytoskeleton of cells is essential for their morphology, migration, and cytokinesis and also for intracellular transport, endo- and exocytosis, as well as a large variety of other processes (Pollard and Borisy 2003). It is comprised of three systems, microtubules, intermediate filaments, and F-actin, each associated with numerous specific accessory and regulatory proteins (Dudek and Garcia 2001). Actin is the central cytoskeletal element of endothelial cells, which comprises up to 15% of total protein content. In confluent endothelial cells, F-actin is found primarily beneath the plasma membrane, forming the pool of “cortical F-actin ” and interacting with cell–cell adhesion complexes through respective adaptor proteins. The presence of this circumferential cortical F-actin ring has been shown to be essential for adhesion in general; if disrupted by Latrunculin A or cytochalasin D, junction integrity is lost (Shen and Turner 2005; Ivanov et al. 2005; Yamazaki et al. 2007; Quinlan and Hyatt 1999).

The formation of cell–cell junctions is usually initiated between migrating cells, which form characteristic lamellipodia with unevenly distributed adhesion molecules at their leading edges (Yap et al. 1997; Hoelzle and Svitkina 2012). This initial assembly of nascent cell–cell contacts is independent of F-actin. However, once this contact is established, the further maturation of junctions relies on an intact cortical actin cytoskeleton (Chu et al. 2006; Chu et al. 2004). Furthermore, linkage to F-actin also influences the strength of the respective intercellular adhesion (Mege et al. 2006). Quickly after initial adhesion site formation at the lamellipodia, the actin-related protein (Arp) 2/3 complex is deactivated, or potentially repressed by an enrichment of α-catenin (Drees et al. 2005; Pokutta and Weis 2007). Consequently, other actin-binding proteins, among them vasodilator-stimulated phosphoprotein) and Mena, are subsequently recruited through α-catenin, leading to the formation of thicker F-actin cables needed to stabilize the developing junctions (Drees et al. 2005; Krause et al. 2003; Scott et al. 2006).

2.1 Adaptor Proteins Link Cortical F-actin to Junctional Proteins

The cortical actin cytoskeleton is crucial for the maintenance of cell–cell junctions. However, many proteins that form intercellular oligomers do not directly interact with F-actin. Therefore, the presence of linker proteins connecting intercellular adhesion proteins to F-actin is especially important.

Prominent examples of such adaptor proteins are α-catenin, VASP, Epithelial protein lost in neoplasm (EPLIN), or afadin, all of which are involved in anchoring adhesion systems to F-actin. Their importance is underlined by different studies, which show how their loss leads to severe defects in junction formation and maintenance (Vasioukhin et al. 2000; Kwiatkowski et al. 2010; Pappas and Rimm 2006; Abe and Takeichi 2008). The underlying reasons for these defects in proper junction formation are varied, depending on the function of the respective protein. Colon carcinoma cell lines deficient in α-catenin were not able to maintain their adhesive properties, due to an inability of forming cadherin-mediated contacts (Pappas and Rimm 2006). VASP activity was shown to be necessary both for F-actin accumulation and its assembly at cell–cell contacts, and sequestering VASP through relocating it to mitochondria led to defects in F-actin organization at contact sites (Scott et al. 2006), while VASP knockdown led to enhanced endothelial permeability (Schlegel et al. 2008; Reinhard et al. 2001). In afadin-depleted epithelial cells, adherens junctions do not form due to defective recruitment of cadherins to initial adherens junction sites, underlining the importance of afadin during initial junction formation (Tachibana et al. 2000). EPLIN is another linker of the cadherin–catenin complex to F-actin and is also needed for the stabilization of cortical actin fibers. Accordingly, siRNA-mediated knockdown of EPLIN in colon adenocarcinoma cell lines led to misorganization of the cortical F-actin, with E-cadherin showing only a punctate accumulation at cell–cell contact zones (Abe and Takeichi 2008; Chervin-Petinot et al. 2012).

2.2 Drebrin Acts as a Stabilizer for Nectins, by Linking Afadin to Cortical F-actin

It is well described that cadherins are linked to the F-actin cytoskeleton. Also, the roles of the different adaptor proteins through which this linkage is established have been explored in depth (Pappas and Rimm 2006; Abe and Takeichi 2008; Tachibana et al. 2000). In contrast, knowledge on how other adherens junction components, in particular nectins , are stabilized in the junctional area is scarce. This is in part based on the fact that nectins are not well characterized in the endothelium in general and are usually only mentioned in the context of initial junction formation (Takahashi et al. 1999; Mizoguchi et al. 2002; Rikitake et al. 2012; Reymond et al. 2000; Ikeda et al. 1999; Mueller et al. 2003). In our work, we could show that drebrin acts by stabilizing the nectin/afadin system through linking it to the cortical F-actin network. These data revealed a novel function of drebrin in the endothelium, as an adaptor protein for the nectin/afadin system (Rehm et al. 2013). In addition, our work showed that nectins are not only necessary for the formation of nascent cell–cell contacts but are also of major importance for the maintenance of established adherens junctions, especially under conditions of enhanced shear stress such as vascular flow (Fig. 21.1).

In our study, we could show that siRNA-mediated depletion of drebrin leads to functional impairments of endothelial monolayers, as demonstrated by a decrease of transendothelial electrical resistance (TER) and also by rupturing of HUVEC monolayers cultured under constant unidirectional flow conditions (Fig. 21.1). The observed weakening of cell–cell contacts upon drebrin depletion is characterized by a specific loss of nectin-2 from adherens junctions, whereas other junctional proteins such as connexin-43, VE-cadherin, occludin, or PECAM-1 remain apparently unaffected (Rehm et al. 2013). Further experiments showed that, in the absence of drebrin, nectin is endocytosed and subsequently degraded in lysosomes, pointing to an important role of drebrin in the stabilization of nectin at the junctional area. The importance of drebrin for nectin’s presence at junctions was further underlined by rescue experiments, where reexpression of siRNA-insensitive drebrin led to recovery of nectin at junctions.

In 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 glutathione S-transferase (GST)-pulldown experiments using bacterially expressed domain constructs of both proteins fused to GST or maltose-binding protein (MBP) (Fig. 21.2). Moreover, confocal microscopy studies revealed strong binding between drebrin and afadin also in a cellular context, as drebrin’s polyproline region fused to a mitochondrial targeting signal is sufficient to relocalize endogenous afadin to the outer membrane of mitochondria. The close association of afadin and drebrin was further demonstrated by FRAP experiments, which showed that afadin’s mobility at the junctional area is enhanced under drebrin depletion. The fact that afadin remains at junctions, in contrast to nectin, is most probably due to accessory binding by other proteins, such as ZO-1 or α-catenin (Tachibana et al. 2000; Takai and Nakanishi 2003).

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, drebrin is able to anchor afadin to the cortical actin cytoskeleton. As afadin can bind to nectin through its PDZ region, this results in a chain of protein interactions, with the sequence F-actin-drebrin-afadin-nectin. Nectin is thus indirectly stabilized by drebrin at junctions through drebrin’s dual ability to bind afadin and F-actin (Fig. 21.3).

Fig. 21.3
figure 3

Drebrin binds afadin and anchors nectin to the F-actin cytoskeleton in endothelial cells. (a) Drebrin binds to F-actin with high affinity through its coiled coil (CC) region and binds via its polyproline (PP) region to afadin’s PR1–2 regions, while afadin’s PDZ domain binds nectin. Nectin is thus stabilized at the junctional region, preserving endothelial integrity . (b) The absence of drebrin and/or afadin leads to the loss of nectin’s indirect anchorage to the actin cytoskeleton . Nectins are subsequently internalized and degraded in lysosomes, resulting in impaired endothelial integrity. (c) Nectin can be artificially stabilized at junctions even in the absence of both afadin and drebrin upon the overexpression of constructs containing the afadin PDZ region and drebrin’s CC region, reestablishing proper anchorage to the actin cytoskeleton

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In order to verify that the linkage of nectin to F-actin is indeed essential for monolayer integrity , we generated a variety of minimal rescue constructs and expressed them in cells under knockdown of both drebrin and afadin. Remarkably, minimal constructs containing only afadin’s PDZ region coupled to drebrin´s F-actin-binding region, or to lifeact, were able to fully rescue nectin at adherens junctions. These results showed that drebrin functions indeed by providing indirect linkage for nectin to F-actin. At the same time, the construct containing lifeact instead of drebrin’s F-actin-binding region demonstrated that only binding to F-actin per se is necessary and that the drebrin-provided anchorage is not based on a specific property of the drebrin–actin interface. Still, drebrin is uniquely equipped to stabilize nectin at endothelial junctions, as it contains binding sites for both afadin and F-actin. This enables drebrin to function as a linker for the nectin/afadin system to the cortical actin cytoskeleton and thus preserve endothelial integrity. Collectively, these results contribute to the current understanding of cell–cell junction regulation in the endothelium, and especially under vascular flow. In particular, the newly identified interaction between drebrin and afadin is shown to be crucial for junctional integrity, not only for junction formation but also during the maintenance of junctions under steady-state conditions (Rehm et al. 2013).