Homophilic interaction and deformation of E-cadherin and cadherin 7 probed by single molecule force spectroscopy

https://doi.org/10.1016/j.abb.2015.10.008Get rights and content

Highlights

  • At the molecular level, the bond between E-cad is stronger than Cad 7.

  • Force-strengthening of homophilic binding of E-cad, but absent in case of Cad 7.

  • Unbinding forces of cadherins overlap with unfolding forces of their EC domains.

  • At a stretching force ∼5 pN, EC domains of E-cad unfold in ∼30 s.

  • Forced-deformation of EC domains is expected to help the strengthening of binding.

Abstract

Cadherin-mediated adhesion plays a crucial role in multicellular organisms. Dysfunction within this adhesion system has major consequences in many pathologies, including cancer invasion and metastasis. However, mechanisms controlling cadherin recognition and adhesive strengthening are only partially understood. Here, we investigated the homophilic interactions and mechanical stability of the extracellular (EC) domains of E-cadherin and cadherin 7 using atomic force microscopy and magnetic tweezers. Besides exhibiting stronger interactions, E-cadherin also showed more efficient force-induced self-strengthening of interactions than cadherin 7. In addition, the distributions of the unbinding forces for both cadherins partially overlap with those of the unfolding forces, indicating that partial unfolding/deformation of the cadherin EC domains may take place during their homophilic interactions. These conformational changes may be involved in cadherins physiology function and contribute to the significant differences in adhesive strength mediated by type I and type II cadherins.

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.

References (60)

  • R. Liu

    Mechanical characterization of protein L in the low-force regime by electromagnetic tweezers/evanescent nanometry

    Biophys. J.

    (2009)
  • J. Wong et al.

    Direct force measurements of the streptavidin-biotin interaction

    Biomol. Eng.

    (1999)
  • M.V. Bayas

    Lifetime measurements reveal kinetic differences between homophilic cadherin bonds

    Biophys. J.

    (2006)
  • J. Oroz

    Nanomechanics of the cadherin ectodomain “CANALIZATION” BYCa2 BINDING results IN A new mechanical element

    J. Biol. Chem.

    (2011)
  • M. Sotomayor et al.

    The allosteric role of the Ca2+ switch in adhesion and elasticity of C-cadherin

    Biophysical J.

    (2008)
  • H. Chen

    Improved high-force magnetic tweezers for stretching and refolding of proteins and short DNA

    Biophysical J.

    (2011)
  • O.J. Harrison

    The extracellular architecture of adherens junctions revealed by crystal structures of type I cadherins

    Structure

    (2011)
  • T.E. Fisher

    The study of protein mechanics with the atomic force microscope

    Trends Biochem. Sci.

    (1999)
  • M. Sotomayor et al.

    In search of the hair-cell gating spring elastic properties of ankyrin and cadherin repeats

    Structure

    (2005)
  • S. Yamada

    Deconstructing the cadherin-catenin-actin complex

    Cell

    (2005)
  • O.J. Harrison

    The extracellular Architecture of Adherens junctions revealed by Crystal structures of type I cadherins

    Structure

    (2011)
  • G.S. Brigidi et al.

    Cadherin-catenin adhesion complexes at the synapse

    Curr. Opin. Neurobiol.

    (2011)
  • T.E. Fisher

    The study of protein mechanics with the atomic force microscope

    Trends Biochem. Sci.

    (1999)
  • B.M. Gumbiner

    Regulation of cadherin-mediated adhesion in morphogenesis

    Nat. Rev. Mol. Cell Biol.

    (2005)
  • R. Umbas

    Expression of the cellular adhesion molecule E-cadherin is reduced or absent in high-grade prostate cancer

    Cancer Res.

    (1992)
  • T.J. Boggon

    C-cadherin ectodomain structure and implications for cell adhesion mechanisms

    Science

    (2002)
  • S. Dufour

    Differential function of N-cadherin and cadherin-7 in the control of embryonic cell motility

    J. Cell Biol.

    (1999)
  • M. Takeichi

    Cadherins: a molecular family important in selective cell-cell adhesion

    Annu. Rev. Biochem.

    (1990)
  • M. Overduin

    Solution structure of the epithelial cadherin domain responsible for selective cell adhesion

    Science

    (1995)
  • Y. Shimoyama

    Identification of three human type-II classic cadherins and frequent heterophilic interactions between different subclasses of type-II classic cadherins

    Biochem. J.

    (2000)
  • Cited by (0)

    1

    Both authors contribute to this paper equally.

    View full text