Homophilic interaction and deformation of E-cadherin and cadherin 7 probed by single molecule force spectroscopy
Introduction
Selective and robust cell–cell adhesion plays a key role in maintaining tissue structural integrity and specific architecture in multicellular organisms [1], [2]. In most tissues, cell–cell adhesion is dominated by a class of transmembrane proteins named cadherins [1], [2]. Dysregulation of cadherin function correlates with tumour cell invasion and distant dissemination [1], [2], [3], [4]. The cadherin superfamily comprises distinct families and subfamilies [5], [6]; one of these is the classical cadherins. E-cadherin, the prototypic member of classical type I cadherins, is an essential component of epithelial adherens junctions and contributes to a fully polarized state in the cell through the formation of a circumferential actin belt. In contrast, classical type II cadherins, such as cadherin 7, show significantly weaker adhesion and are mainly expressed in mesenchymal tissues [6], [7].
Type I and type II cadherins demonstrate similar domain organization: a cytoplasmic region, a transmembrane region, and an extracellular region [8], [9]. The primary sequence of the extracellular region differs significantly between type I and type II cadherins [10]. The forces required to separate cell doublets expressing type II cadherins are much weaker than for those of type I-expressing cells, a property linked to their extracellular region [6]. Nonetheless, the extracellular segments of type I and type II cadherins share a similar 3D structure that comprises five tandem repeats, called extracellular cadherin (EC) domains, herein referred to as EC1 to EC5. Each EC domain consists of about 110 amino acids forming seven β-strands that are organized into two β-sheets [5], [11], [12].
Crystallographic data suggest the formation of X-dimers and strand-swapping dimers by the homophilic interaction of classical type I cadherins in vitro [12], [13], [14]. In a two-step adhesive binding experiment, cadherins were shown to initially form X-dimers and then convert to strand-swapping dimers [12]. A similar pathway were also proposed by Rakshit et al., as in the atomic force microscopy (AFM) studies [15], they found that strand-swapping dimers formed slip bonds, and X-dimer of E-cadherin formed catch bonds [15]. The latest steered molecular dynamics simulations results suggest that tensile force can deform cadherin EC domains to form long-lived hydrogen bonds to tighten the X-dimer contact [16]. Crystallographic studies also show that type II cadherins form similar strand-swap dimers [13], [14]. In their strand-swapping dimers, the buried accessible surface area was found larger than that of type I cadherins [13], [14] and, the dissociation constants (kd) measured by ultracentrifugation [17] imply that the binding energy of type II cadherins is higher than that of type I cadherins. On the contrary, type I cadherins expressed cells show stronger unbinding forces [6], [7]. Nevertheless, direct comparison between type I and type II cadherins at the molecular level is lacking, while this is important for understanding the distinct adhesion mechanism between them.
In the AFM study of E-cadherin X-dimers and strand-swapping dimers, Rakshit et al. proposed a model of reorientation of the EC domains by tensile forces to lock the dimer more tightly by an alternate binding site as the mechanism of the catch-bond behaviour. Meanwhile, quite a few studies also indicate that force plays important role in assisting cadherin-mediated adhesion processes. E-cadherin-mediated adhesion occurs under an actomyosin-generated tension force in vivo [18], force can enhance E-cadherin-mediated adhesion [19], [20], [21], and force can also increase the junction size in cadherin adhesions [22], [23]. In addition, studies indicated that the cells can respond to the activation of E-cadherin EC domains (conformational change for binding) to regulate adhesion [24]. Therefore, EC domains and the homophilic interactions of their pairs response to mechanical forces is essential for cell–cell interaction.
Here, we used AFM to compare the homophilic interactions between E-cadherin and cadherin 7 at the single-molecule level varied under the external force dynamics. While both cadherins showed slightly time-dependent strengthening in their homophilic interactions, the strengthening effect by additional mechanical stretching is much more noticeable for E-cadherin than for cadherin 7. The elasticity of the EC domains of both cadherins were also carried out using AFM and magnetic tweezers, and the results indicated that the force to partially unfold/deform the EC domains showed a larger overlap with the unbinding force of the dimers for E-cadherin than cadherin 7.
Section snippets
Protein cloning
The Ecad and Cad7 genes were cloned into pFB-Sec-NH vector (Addgene) using ligation-independent cloning [25]. The forward and reverse primers for Cad7 were 5’ –TACTTCCAATCCATGAGCTGGGTTTGGAATCAGTTC-3′ and 5′- TATCCACCTTTACTGTCACTCTGCATTGCAGGTCTGG-3′, and for Ecad, 5′-TACTTCCAATCCATGGACTGGGTCATCCCTCCC-3′ and 5′-TATCCACCTTTACTGTCACGCCTTCATGCAGTTGTTGA-3’. The construct contains baculovirus gp64 signal peptide followed by an N-terminal hexahistidine tag and TEV protease cleavage site. Bacmid
Results
AFM was used to measure the unbinding forces of homophilic interaction pairs between E-cadherin and cadherin 7 EC domains. The molecular elasticity of these EC domains was also investigated using AFM and magnetic tweezers; in the latter case, the stretching forces can be as low as a few pN, which is close to the in vivo forces borne by adhesion molecules.
The binding force of E-cadherin is stronger than cadherin 7
The AFM results show that the interaction between E-cadherin (type I) is stronger than that between cadherin 7 (type II), as the average unbinding force is higher for E-cadherins (Fig. 2). This is in agreement with in vivo cell adhesion measurements [6], [7]. However, here AFM experiments are measuring the unbinding force at single-molecule level, so strengthening mechanisms at the cellular level, e.g. lateral clusters [43], [44] and other cytoplasmic mechanosensing proteins [19], [20], [21],
Acknowledgements
We gratefully acknowledge support from the Research Start Fund for Talent Recruitment, Chongqing University, China, and the seed grant (WBS R-714-002-007-271) from the Mechanobiology Institute, Singapore.
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Both authors contribute to this paper equally.